RBPMS2 is a conserved regulator of alternative splicing that promotes myofibrillar organization and optimal calcium handling in cardiomyocytes
PMID: 34767451 DOI:10.1126/sciadv.abk0271
Stem cells are remarkably small. Whether small size is important for stem cell function is unknown. We find that hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferation and was accompanied by altered metabolism. Preventing HSC enlargement or reducing large HSCs in size averts the loss of stem cell potential under conditions causing stem cell exhaustion. Last, we show that murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.
PMID: 33972784 DOI: 10.1038/s41594-021-00589-3
RNA polymerase II (RNAPII) pausing immediately downstream of the transcription start site is a critical rate-limiting step for the expression of most metazoan genes. During pause release, RNAPII encounters a highly conserved +1 H2A.Z nucleosome, yet how this histone variant contributes to transcription is poorly understood. Here, using an inducible protein degron system combined with genomic approaches and live cell super-resolution microscopy, we show that H2A.Z.1 modulates RNAPII dynamics across most genes in murine embryonic stem cells. Our quantitative analysis shows that H2A.Z.1 slows the rate of RNAPII pause release and consequently impacts negative elongation factor dynamics as well as nascent transcription. Consequently, H2A.Z.1 also impacts re-loading of the pre-initiation complex components TFIIB and TBP. Altogether, this work provides a critical mechanistic link between H2A.Z.1 and the proper induction of mammalian gene expression programs through the regulation of RNAPII dynamics and pause release.
PMID: 32939090 DOI:10.1038/s41586-020-2732-8
Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.
Cheng CW, Biton M, Haber AL, Gunduz N, Eng G, Gaynor LT, Tripathi S, Calibasi-Kocal G, Rickelt S, Butty VL, Moreno-Serrano M, Iqbal AM, Bauer-Rowe KE, Imada S, Ulutas MS, Mylonas C, Whary MT, Levine SS, Basbinar Y, Hynes RO, Mino-Kenudson M, Deshpande V, Boyer LA, Fox JG, Terranova C, Rai K, Piwnica-Worms H, Mihaylova MM, Regev A, Yilmaz ÖH.
Cell. 2019 Aug 22;178(5):1115-1131.e15.
Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (βOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes βOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous βOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, βOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through βOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of βOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.
Ben-Yair R, Butty VL, Busby M, Qiu Y, Levine SS, Goren A, Boyer LA, Burns CG, Burns CE.
Development. 2019 Aug 19. pii: dev.178632.
Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms, such as zebrafish, capable of robust cardiac renewal represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following injury. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3K27M that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprised of H3.3K27M-expressing cardiomyocytes fail to regenerate with wound edge cells showing heightened expression of structural genes and prominent sarcomere structures. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of infarcted human hearts might improve its regenerative capacity.
Failed progenitor specification underlies the cardiopharyngeal phenotypes in a zebrafish model of 22q11.2 deletion syndrome.
Guner-Ataman B, González-Rosa JM, Shah HN, Butty VL, Jeffrey S, Abrial M, Boyer LA, Burns CG, Burns CE.
Cell Rep. 2018 Jul 31;24(5):1342-1354.
Microdeletions involving TBX1 result in variable congenital malformations known collectively as 22q11.2 deletion syndrome (22q11.2DS). Tbx1-deficient mice and zebrafish recapitulate several disease phenotypes, including pharyngeal arch artery (PAA), head muscle (HM), and cardiac outflow tract (OFT) deficiencies. In zebrafish, these structures arise from nkx2.5+ progenitors in pharyngeal arches 2–6. Because pharyngeal arch morphogenesis is compromised in Tbx1-deficient animals, the malformations were considered secondary. Here, we report that the PAA, HM, and OFT phenotypes in tbx1 mutant zebrafish are primary and arise prior to pharyngeal arch morphogenesis from failed specification of the nkx2.5+ pharyngeal lineage. Through in situ analysis and lineage tracing, we reveal that nkx2.5 and tbx1 are co-expressed in this progenitor population. Furthermore, we present evidence suggesting that gdf3-ALK4 signaling is a downstream mediator of nkx2.5+ pharyngeal lineage specification. Collectively, these studies support a cellular mechanism potentially underlying the cardiovascular and cranio-facial defects observed in the 22q11.2DS population.
Edington CD, Chen WLK, Geishecker E, Kassis T, Soenksen LR, Bhushan BM, Freake D, Kirschner J, Maass C, Tsamandouras N, Valdez J, Cook CD, Parent T, Snyder S, Yu J, Suter E, Shockley M, Velazquez J, Velazquez JJ, Stockdale L, Papps JP, Lee I, Vann N, Gamboa M, LaBarge ME, Zhong Z, Wang X, Boyer LA, Lauffenburger DA, Carrier RL, Communal C, Tannenbaum SR, Stokes CL, Hughes DJ, Rohatgi G, Trumper DL, Cirit M, Griffith LG.
Sci Rep. 2018 Mar 14;8(1):4530.
Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs – “4-way”, “7-way”, and “10-way” – each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS “physiome-on-a-chip” approaches in drug discovery.
Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing
Werley CA, Chien MP, Gaublomme J, Shekhar K, Butty V, Yi BA, Kralj JM, Bloxham W, Boyer LA, Regev A, Cohen AE.
PLoS One. 2017 Mar 23;12(3):e0172671.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation.
Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units.
Uzel SG, Platt RJ, Subramanian V, Pearl TM, Rowlands CJ, Chan V, Boyer LA, So PT, Kamm RD.
Sci Adv. 2016 Aug 3;2(8):e1501429
Motor units are the fundamental elements responsible for muscle movement. They are formed by lower motor neurons and their muscle targets, synapsed via neuromuscular junctions (NMJs). The loss of NMJs in neurodegenerative disorders (such as amyotrophic lateral sclerosis or spinal muscle atrophy) or as a result of traumatic injuries affects millions of lives each year. Developing in vitro assays that closely recapitulate the physiology of neuromuscular tissues is crucial to understand the formation and maturation of NMJs, as well as to help unravel the mechanisms leading to their degeneration and repair. We present a microfluidic platform designed to coculture myoblast-derived muscle strips and motor neurons differentiated from mouse embryonic stem cells (ESCs) within a three-dimensional (3D) hydrogel. The device geometry mimics the spinal cord-limb physical separation by compartmentalizing the two cell types, which also facilitates the observation of 3D neurite outgrowth and remote muscle innervation. Moreover, the use of compliant pillars as anchors for muscle strips provides a quantitative functional readout of force generation. Finally, photosensitizing the ESC provides a pool of source cells that can be differentiated into optically excitable motor neurons, allowing for spatiodynamic, versatile, and noninvasive in vitro control of the motor units.
van der Harst P, van Setten J, Verweij N, Vogler G, Franke L, Maurano MT, Wang X, Mateo Leach I, Eijgelsheim M, Sotoodehnia N, Hayward C, Sorice R, Meirelles O, Lyytikäinen LP, Polašek O, Tanaka T, Arking DE, Ulivi S, Trompet S, Müller-Nurasyid M, Smith AV, Dörr M, Kerr KF, Magnani JW, Del Greco M F, Zhang W, Nolte IM, Silva CT, Padmanabhan S, Tragante V, Esko T, Abecasis GR, Adriaens ME, Andersen K, Barnett P, Bis JC, Bodmer R, Buckley BM, Campbell H, Cannon MV, Chakravarti A, Chen LY, Delitala A, Devereux RB, Doevendans PA, Dominiczak AF, Ferrucci L, Ford I, Gieger C, Harris TB, Haugen E, Heinig M, Hernandez DG, Hillege HL, Hirschhorn JN, Hofman A, Hubner N, Hwang SJ, Iorio A, Kähönen M, Kellis M, Kolcic I, Kooner IK, Kooner JS, Kors JA, Lakatta EG, Lage K, Launer LJ, Levy D, Lundby A, Macfarlane PW, May D, Meitinger T, Metspalu A, Nappo S, Naitza S, Neph S, Nord AS, Nutile T, Okin PM, Olsen JV, Oostra BA, Penninger JM, Pennacchio LA, Pers TH, Perz S, Peters A, Pinto YM, Pfeufer A, Pilia MG, Pramstaller PP, Prins BP, Raitakari OT, Raychaudhuri S, Rice KM, Rossin EJ, Rotter JI, Schafer S, Schlessinger D, Schmidt CO, Sehmi J, Silljé HHW, Sinagra G, Sinner MF, Slowikowski K, Soliman EZ, Spector TD, Spiering W, Stamatoyannopoulos JA, Stolk RP, Strauch K, Tan ST, Tarasov KV, Trinh B, Uitterlinden AG, van den Boogaard M, van Duijn CM, van Gilst WH, Viikari JS, Visscher PM, Vitart V, Völker U, Waldenberger M, Weichenberger CX, Westra HJ, Wijmenga C, Wolffenbuttel BH, Yang J, Bezzina CR, Munroe PB, Snieder H, Wright AF, Rudan I, Boyer LA, Asselbergs FW, van Veldhuisen DJ, Stricker BH, Psaty BM, Ciullo M, Sanna S, Lehtimäki T, Wilson JF, Bandinelli S, Alonso A, Gasparini P, Jukema JW, Kääb S, Gudnason V, Felix SB, Heckbert SR, de Boer RA, Newton-Cheh C, Hicks AA, Chambers JC, Jamshidi Y, Visel A, Christoffels VM, Isaacs A, Samani NJ, de Bakker PIW.
J Am Coll Cardiol. 2016 Sep 27;68(13):1435-1448.
Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death.
A G-Rich Motif in the lncRNA Braveheart Interacts with a Zinc-Finger Transcription Factor to Specify the Cardiovascular Lineage.
Xue Z, Hennelly S, Doyle B, Gulati AA, Novikova IV, Sanbonmatsu KY, Boyer LA.
Mol Cell. 2016 Oct 6;64(1):37-50.
Long non-coding RNAs (lncRNAs) are an emerging class of transcripts that can modulate gene expression; however, their mechanisms of action remain poorly understood. Here, we experimentally determine the secondary structure of Braveheart (Bvht) using chemical probing methods and show that this ∼590 nt transcript has a modular fold. Using CRISPR/Cas9-mediated editing of mouse embryonic stem cells, we find that deletion of 11 nt in a 5′ asymmetric G-rich internal loop (AGIL) of Bvht (bvhtdAGIL) dramatically impairs cardiomyocyte differentiation. We demonstrate a specific interaction between AGIL and cellular nucleic acid binding protein (CNBP/ZNF9), a zinc-finger protein known to bind single-stranded G-rich sequences. We further show that CNBP deletion partially rescues the bvhtdAGIL mutant phenotype by restoring differentiation capacity. Together, our work shows that Bvht functions with CNBP through a well-defined RNA motif to regulate cardiovascular lineage commitment, opening the door for exploring broader roles of RNA structure in development and disease.
Smith JG, Felix JF, Morrison AC, Kalogeropoulos A, Trompet S, Wilk JB, Gidlöf O, Wang X, Morley M, Mendelson M, Joehanes R, Ligthart S, Shan X, Bis JC, Wang YA, Sjögren M, Ngwa J, Brandimarto J, Stott DJ, Aguilar D, Rice KM, Sesso HD, Demissie S, Buckley BM, Taylor KD, Ford I, Yao C, Liu C; CHARGE-SCD consortium; EchoGen consortium; QT-IGC consortium; CHARGE-QRS consortium, Sotoodehnia N, van der Harst P, Stricker BH, Kritchevsky SB, Liu Y, Gaziano JM, Hofman A, Moravec CS, Uitterlinden AG, Kellis M, van Meurs JB, Margulies KB, Dehghan A, Levy D, Olde B, Psaty BM, Cupples LA, Jukema JW, Djousse L, Franco OH, Boerwinkle E, Boyer LA, Newton-Cheh C, Butler J, Vasan RS, Cappola TP, Smith NL.
PLoS Genet. 2016 May 5;12(5):e1006034.
Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinants of mortality in patients with new-onset heart failure, we performed a meta-analysis of genome-wide association studies and follow-up genotyping in independent populations. We identified and replicated an association for a genetic variant on chromosome 5q22 with 36% increased risk of death in subjects with heart failure (rs9885413, P = 2.7×10-9). We provide evidence from reporter gene assays, computational predictions and epigenomic marks that this polymorphism increases activity of an enhancer region active in multiple human tissues. The polymorphism was further reproducibly associated with a DNA methylation signature in whole blood (P = 4.5×10-40) that also associated with allergic sensitization and expression in blood of the cytokine TSLP (P = 1.1×10-4). Knockdown of the transcription factor predicted to bind the enhancer region (NHLH1) in a human cell line (HEK293) expressing NHLH1 resulted in lower TSLP expression. In addition, we observed evidence of recent positive selection acting on the risk allele in populations of African descent. Our findings provide novel genetic leads to factors that influence mortality in patients with heart failure.
Discovery and validation of sub-threshold genome-wide association study loci using epigenomic signatures.
Wang X, Tucker NR, Rizki G, Mills R, Krijger PH, de Wit E, Subramanian V, Bartell E, Nguyen XX, Ye J, Leyton-Mange J, Dolmatova EV, van der Harst P, de Laat W, Ellinor PT, Newton-Cheh C, Milan DJ, Kellis M, Boyer LA.
Elife. 2016 May 10;5.
Genetic variants identified by genome-wide association studies explain only a modest proportion of heritability, suggesting that meaningful associations lie ‘hidden’ below current thresholds. Here, we integrate information from association studies with epigenomic maps to demonstrate that enhancers significantly overlap known loci associated with the cardiac QT interval and QRS duration. We apply functional criteria to identify loci associated with QT interval that do not meet genome-wide significance and are missed by existing studies. We demonstrate that these ‘sub-threshold’ signals represent novel loci, and that epigenomic maps are effective at discriminating true biological signals from noise. We experimentally validate the molecular, gene-regulatory, cellular and organismal phenotypes of these sub-threshold loci, demonstrating that most sub-threshold loci have regulatory consequences and that genetic perturbation of nearby genes causes cardiac phenotypes in mouse. Our work provides a general approach for improving the detection of novel loci associated with complex human traits.
Surface LE, Fields PA, Subramanian V, Behmer R, Udeshi N, Peach SE, Carr SA, Jaffe JD, Boyer LA.
Cell Rep. 2016 Feb 9;14(5):1142-1155.
Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in embryonic stem cells (ESCs) to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 monoubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1(K3R3)). We show that H2A.Z.1(K3R3) is properly incorporated at target promoters in murine ESCs (mESCs), but loss of monoubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member BRD2, are enriched in H2A.Z.1 chromatin. We further show that BRD2 is gained at de-repressed promoters in H2A.Z.1(K3R3) mESCs, whereas BRD2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and BRD2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs.
Verweij N, Mateo Leach I, Isaacs A, Arking DE, Bis JC, Pers TH, Van Den Berg ME, Lyytikäinen LP, Barnett P, Wang X; LifeLines Cohort Study, Soliman EZ, Van Duijn CM, Kähönen M, Van Veldhuisen DJ, Kors JA, Raitakari OT, Silva CT, Lehtimäki T, Hillege HL, Hirschhorn JN, Boyer LA, Van Gilst WH, Alonso A, Sotoodehnia N, Eijgelsheim M, De Boer RA, De Bakker PI, Franke L, Van Der Harst P.
Hum Mol Genet. 2016 May 15;25(10):2093-2103.
The ST-segment and adjacent T-wave (ST-T wave) amplitudes of the electrocardiogram are quantitative characteristics of cardiac repolarization. Repolarization abnormalities have been linked to ventricular arrhythmias and sudden cardiac death. We performed the first genome-wide association meta-analysis of ST-T-wave amplitudes in up to 37 977 individuals identifying 71 robust genotype-phenotype associations clustered within 28 independent loci. Fifty-four genes were prioritized as candidates underlying the phenotypes, including genes with established roles in the cardiac repolarization phase (SCN5A/SCN10A, KCND3, KCNB1, NOS1AP and HEY2) and others with as yet undefined cardiac function. These associations may provide insights in the spatiotemporal contribution of genetic variation influencing cardiac repolarization and provide novel leads for future functional follow-up.
O’Meara CC, Wamstad JA, Gladstone RA, Fomovsky GM, Butty VL, Shrikumar A, Gannon JB, Boyer LA, Lee RT.
Circ Res. 2015 Feb 27;116(5):804-15.
We derived a core transcriptional signature of injury-induced cardiac myocyte (CM) regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo CM differentiation, in vitro CM explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of CM differentiation processes, including reactivation of latent developmental programs similar to those observed during destabilization of a mature CM phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13, which induced CM cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of interleukin 13 signaling in CMs. These downstream signaling molecules are also modulated in the regenerating mouse heart.
Subramanian V, Fields PA, Boyer LA.
F1000Prime Rep. 2015 Jan 5;7:01.
O’Meara CC, Wamstad JA, Gladstone RA, Fomovsky GM, Butty VL, Shrikumar A, Gannon JB, Boyer LA, Lee RT.
The replacement of nucleosomal H2A with the histone variant H2A.Z is critical for regulating DNA-mediated processes across eukaryotes and for early development of multicellular organisms. How this variant performs these seemingly diverse roles has remained largely enigmatic. Here, we discuss recent mechanistic insights that have begun to reveal how H2A.Z functions as a molecular rheostat for gene control. We focus on specific examples in metazoans as a model for understanding how H2A.Z integrates information from histone post-translational modifications, other histone variants, and transcription factors (TFs) to regulate proper induction of gene expression programs in response to cellular cues. Finally, we propose a general model of how H2A.Z incorporation regulates chromatin states in diverse processes.
Rege M, Subramanian V, Zhu C, Hsieh TH, Weiner A, Friedman N, Clauder-Münster S, Steinmetz LM, Rando OJ, Boyer LA, Peterson CL.
Cell Rep. 2015 Nov 24;13(8):1610-22.
The histone variant H2A.Z is a hallmark of nucleosomes flanking promoters of protein-coding genes and is often found in nucleosomes that carry lysine 56-acetylated histone H3 (H3-K56Ac), a mark that promotes replication-independent nucleosome turnover. Here, we find that H3-K56Ac promotes RNA polymerase II occupancy at many protein-coding and noncoding loci, yet neither H3-K56Ac nor H2A.Z has a significant impact on steady-state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on RNA levels are revealed only in the absence of the nuclear RNA exosome. H2A.Z is also necessary for the expression of divergent, promoter-proximal noncoding RNAs (ncRNAs) in mouse embryonic stem cells. Finally, we show that H2A.Z functions with H3-K56Ac to facilitate formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher-order chromatin structures.
Rizki G, Boyer LA.
Circ Res. 2015 Jul 3;117(2):192-206.
Transcriptional and epigenetic regulation is critical for proper heart development, cardiac homeostasis, and pathogenesis. Long noncoding RNAs have emerged as key components of the transcriptional regulatory pathways that govern cardiac development as well as stress response, signaling, and remodeling in cardiac pathologies. Within the past few years, studies have identified many long noncoding RNAs in the context of cardiovascular biology and have begun to reveal the key functions of these transcripts. In this review, we discuss the growing roles of long noncoding RNAs in different aspects of cardiovascular development as well as pathological responses during injury or disease. In addition, we discuss diverse mechanisms by which long noncoding RNAs orchestrate cardiac transcriptional programs. Finally, we explore the exciting potential of this novel class of transcripts as biomarkers and novel therapeutic targets for cardiovascular diseases.
Roadmap Epigenomics Consortium, Kundaje A, Meuleman W, Ernst J, Bilenky M, Yen A, Heravi-Moussavi A, Kheradpour P, Zhang Z, Wang J, Ziller MJ, Amin V, Whitaker JW, Schultz MD, Ward LD, Sarkar A, Quon G, Sandstrom RS, Eaton ML, Wu YC, Pfenning AR, Wang X, Claussnitzer M, Liu Y, Coarfa C, Harris RA, Shoresh N, Epstein CB, Gjoneska E, Leung D, Xie W, Hawkins RD, Lister R, Hong C, Gascard P, Mungall AJ, Moore R, Chuah E, Tam A, Canfield TK, Hansen RS, Kaul R, Sabo PJ, Bansal MS, Carles A, Dixon JR, Farh KH, Feizi S, Karlic R, Kim AR, Kulkarni A, Li D, Lowdon R, Elliott G, Mercer TR, Neph SJ, Onuchic V, Polak P, Rajagopal N, Ray P, Sallari RC, Siebenthall KT, Sinnott-Armstrong NA, Stevens M, Thurman RE, Wu J, Zhang B, Zhou X, Beaudet AE, Boyer LA, De Jager PL, Farnham PJ, Fisher SJ, Haussler D, Jones SJ, Li W, Marra MA, McManus MT, Sunyaev S, Thomson JA, Tlsty TD, Tsai LH, Wang W, Waterland RA, Zhang MQ, Chadwick LH, Bernstein BE, Costello JF, Ecker JR, Hirst M, Meissner A, Milosavljevic A, Ren B, Stamatoyannopoulos JA, Wang T, Kellis M.
Nature. 2015 Feb 19;518(7539):317-30.
The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.
Polycomb Repressive Complex 2 regulates lineage fidelity during embryonic stem cell differentiation.
Thornton SR, Butty VL, Levine SS, Boyer LA.
PLoS One. 2014 Oct 21;9(10):e110498.
Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, making it difficult to determine the precise roles of PRC2 during lineage commitment. Moreover, while studies suggest that PRC2 prevents DNA methylation, how these two epigenetic regulators coordinate to regulate lineage programs is poorly understood. Using several PRC2 mutant ESC lines that maintain varying levels of H3K27me3, we found that partial maintenance of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). In contrast, genes that function to specify other lineages failed to be repressed in these cells, suggesting that PRC2 is also necessary for lineage fidelity. We also found that loss of H3K27me3 leads to a modest gain in DNA methylation at PRC2 target regions in both ESCs and in SMNs. Our study demonstrates a critical role for PRC2 in safeguarding lineage decisions and in protecting genes against inappropriate DNA methylation.
Wamstad JA, Wang X, Demuren OO, Boyer LA.
Trends Cell Biol. 2014 May;24(5):294-302.
Advances in genome research have provided an unprecedented opportunity to investigate the function of non-coding DNA regulatory regions that control transcription. Large-scale studies have recently identified hundreds of thousands of distal enhancer elements; their discovery has revealed new insights into the mechanistic details of how tissue-specific gene expression patterns are established and maintained during development. Emerging evidence indicates that lineage-specific transcription factors and chromatin regulators coordinate the activation of distal enhancers to ensure robust control of gene expression programs in a cell type-specific manner. We discuss recent progress in the field and emphasize examples related to the cardiac lineage, where possible, as a model for understanding the contribution of enhancer biology to development and how disruption of enhancer function leads to disease.
H2A.Z acidic patch couples chromatin dynamics to regulation of gene expression programs during ESC differentiation.
Subramanian V, Mazumder A, Surface LE, Butty VL, Fields PA, Alwan A, Torrey L, Thai KK, Levine SS, Bathe M, Boyer LA.
PLoS Genet. 2013;9(8):e1003725.
The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z(AP3)) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z(AP3) interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z(AP3) was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z(AP3) ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z(AP3) ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z(AP3) displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z(AP3) mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.
Scheuermann JC, Boyer LA.
EMBO J. 2013 Jul 3;32(13):1805-16.
Cardiogenesis in mammals requires exquisite control of gene expression and faulty regulation of transcriptional programs underpins congenital heart disease (CHD), the most common defect among live births. Similarly, many adult cardiac diseases involve transcriptional changes and sometimes have a developmental basis. Long non-coding RNAs (lncRNAs) are a novel class of transcripts that regulate cellular processes by controlling gene expression; however, detailed insights into their biological and mechanistic functions are only beginning to emerge. Here, we discuss recent findings suggesting that lncRNAs are important factors in regulation of mammalian cardiogenesis and in the pathogenesis of CHD as well as adult cardiac disease. We also outline potential methodological and conceptual considerations for future studies of lncRNAs in the heart and other contexts.
SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state.
Lodato MA, Ng CW, Wamstad JA, Cheng AW, Thai KK, Fraenkel E, Jaenisch R, Boyer LA.
PLoS Genet. 2013;9(2):e1003288.
SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors.
Klattenhoff CA, Scheuermann JC, Surface LE, Bradley RK, Fields PA, Steinhauser ML, Ding H, Butty VL, Torrey L, Haas S, Abo R, Tabebordbar M, Lee RT, Burge CB, Boyer LA.
Cell. 2013 Jan 31;152(3):570-83.
Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.
Wamstad JA, Alexander JM, Truty RM, Shrikumar A, Li F, Eilertson KE, Ding H, Wylie JN, Pico AR, Capra JA, Erwin G, Kattman SJ, Keller GM, Srivastava D, Levine SS, Pollard KS, Holloway AK, Boyer LA, Bruneau BG.
Cell. 2012 Sep 28;151(1):206-20.
Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel preactivation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease.
Surface LE, Thornton SR, Boyer LA.
Cell Stem Cell. 2010 Sep 3;7(3):288-98.
Precise control of gene expression patterns is critical for the specification of cellular diversity during metazoan development. Polycomb group (PcG) proteins comprise a class of transcriptional modifiers that have dynamic and essential roles in regulating a number of key processes including lineage commitment. How this is accomplished during mammalian development is incompletely understood. Here, we discuss recent studies in embryonic stem cells (ESCs) that provide critical new insights into how PcG proteins may be targeted to genomic sites as well as the mechanisms by which these regulators influence gene expression and multilineage differentiation in mammals.
Sha K, Boyer LA.
StemBook [Internet]. Cambridge (MA): Harvard Stem Cell Institute; 2008-.
2009 May 31.
Pluripotency, a property ascribed to the cells that constitute the early embryo as well as to embryonic stem (ES) cells, is progressively lost during differentiation as the activation of lineage-specific programs steer the cell towards a particular fate. The regulation of chromatin structure has emerged as a key mechanism to modulate developmental gene expression patterns by contributing to the activation or silencing of subsets of genes and through maintenance of expression states during subsequent cell divisions. Recent global analyses have revealed key differences in the chromatin landscape in pluripotent embryonic stem (ES) cells as compared to lineage-committed cells, suggesting that chromatin states may be linked to cell fate. Moreover, the molecular and biochemical characterization of a large group of enzymes that regulate chromatin structure and organization has revealed roles for these factors in early development and stem cell function. Such studies have begun to provide critical insights into how the ES cell genome may remain uncommitted, yet poised for differentiation. These findings have broad implications for understanding development as well as the process of re-programming the somatic genome into a pluripotent-like state and of the progression from normal to disease states.
Subramanian V, Klattenhoff CA, Boyer LA.
Cell Stem Cell. 2009 May 8;4(5):377-8.
Two recent studies, including one in this issue of Cell Stem Cell, have identified novel regulators of embryonic stem cell (ESC) self-renewal using genome-wide RNAi screens in mouse ESCs (Ding et al., 2009; Hu et al., 2009) and have further expanded the repertoire of factors that regulate ESC identity.
Complete list of published work can be found here.