Post-mitotic gene reactivation
A key question in the development of cellular lineages is how transcriptional programs are stably maintained throughout the cell cycle to preserve lineage identity. This question is intimately related to how transcription factors interact with the appropriate gene-specific elements within chromatin and how these interactions are controlled throughout the cell division cycle. During mitosis chromosomes condense and transcription is silenced globally as a result of eviction of most nuclear factors from chromatin. Our studies are aimed at understanding how the cell “remembers” to restore appropriate transcription patterns upon G1 entry. By studying the tri-thorax protein MLL and the hematopoietic transcription factor GATA1 we have gained important insights into mitotic “bookmarking” mechanisms, including the first genome wide location analyses of transcription factors in pure mitotic populations (Blobel et al., 2009; Kadauke et al., 2012) and functional insights into post-mitotic reactivation of bookmarked vs non-bookmarked genes. In the process we developed key reagents that are being used by many others in the field, including a new method to purify mitotic cells to virtual homogeneity and new tools to degrade proteins of interest specifically in mitosis. We are now pursuing studies on how GATA1 is retained at some of its sites during mitosis but not others. We are asking whether mitotic retention of MLL translates to MLL translocation fusion proteins involved in leukemias, and whether mitotic memory is maintained or perturbed in MLL leukemia. Finally, we have carried out the first genome wide survey of chromatin accessibility in pure mitotic chromatin and found that remarkably, that chromosome retain most of their “openness” with enhancers being more susceptible to partial loss of accessibility that promoters (Hsiung et al., 2015). We are examining the mechanisms of transcription reactivation following mitosis.
Publications
Blobel GA, Kadauke S, Wang E, Lau AW, Zuber J, Chou MM, and Vakoc CR (2009). A Reconfigured Pattern of MLL Occupancy within Mitotic Chromatin Promotes Rapid Transcriptional Reactivation Following Mitotic Exit. Molecular Cell, 36:970-983. [PMCID: PMC2818742] (Preview in Developmental Cell 18:4, 2010).
Kadauke S, Udugama M., Pawlicki JM, Achtman JC, Jain DP, Cheng Y, Hardison RC, and Blobel GA (2012) Tissue-specific Mitotic Bookmarking by Hematopoietic Transcription Factor GATA1. Cell 150:725-737. [PMCID: PMC3425057]
Hsiung CC-S, Morrissey C, Udugama M, Frank CL, Keller CA, Baek S, Giardine B, Crawford GE, Sung M-H, Hardison RC, Blobel GA (2015) Genome accessibility is widely preserved and locally modulated during mitosis. Genome Research, 2:213-25
Hsiung CC-S, Keller CA, Ginart P, Jahn KS, Bartman C, Stonestrom AJ, Evans P, Giardine B, Hardison RC, Raj A, Blobel GA (2015) A hyperactive transcriptional state marks genome reactivation upon mitotic exit. Submitted.
Forced chromatin looping
We are pursing questions of how chromatin in organized in the nucleus, specifically, how enhancer-promoter contacts are formed. We found that the hematopoietic transcription factor GATA1 and its co-factor FOG1 are essential to juxtapose the enhancer of the b-globin locus with the promoter (Vakoc et al., 2005). This study was among the first to define any nuclear factor in chromatin looping. We discovered that chromatin looping is highly dynamic and can occur even at repressed genes (Jing et al., Mol. Cell 2008). Using a novel approach of tethering candidate looping factors to an endogenous gene, we were able for the first time to generate an enhancer-promoter chromatin loop at a native endogenous gene locus and thus discovered that chromatin looping causally underlies gene expression (Deng et al., Cell 2012). Subsequently, we adapted this approach to reprogram the murine and human b-globin loci to reactivate the dormant embryonic and fetal globin genes, respectively (Deng et al. 2014). Our long term goal is to advance this strategy towards a clinical application in the setting of sickle cell anemia and thalassemia.
Publications
Vakoc CR, Letting DL, Gheldof N, Sawado T, Bender MA, Groudine M, Weiss MJ, Dekker J, Blobel GA. Proximity among distant regulatory elements at the gamma-globin locus requires GATA-1 and FOG-1. Molecular Cell 17:453-462, 2005. PMID: 15694345
Jing H, Vakoc CR, Mandat S, Wang H, Zheng X, and Blobel GA (2008). Exchange of GATA factors mediates transitions in looped chromatin organization at a developmentally regulated gene locus. Molecular Cell, 29:232-242. [PMCID: PMC2254447] (Preview in Molecular Cell 29:154, 2008)
Deng W, Lee J, Wang H, Reik A, Gregory PD, Dean A, and Blobel GA (2012) Controlling long-range chromosomal interactions at a native locus by targeted tethering of a looping factor. Cell 149:1233-44. (Featured in a Cell video, and selected by Faculty of 1000) [PMCID: PMC3600827]
Deng W, Rupon JW, Krivega, I, Breda L, Motta, I, Jahn KS, Reik A, Gregory PD, Rivella S, Dean A, Blobel GA (2014) Reactivation of developmentally silenced globin gene expression by forced chromatin looping. Cell, 158:849-60. Selected by Faculty of 1000, and highlighted in Nature Genetics (PMCID:PMC4134511)
Bartman C, Hsiung CC-S, Raj A, Blobel GA (2015) Enhancer regulation of transcriptional bursting parameters revealed by forced chromatin looping. Submitted.
Lee K, Hsiung CC-S, Huang P, Raj A, Blobel GA (2015) Dynamic enhancer-gene body contacts during transcription elongation. Genes Dev., in press.
Erythroid transcriptional regulation
Another focus of our laboratory is to understand how transcription factors control hematopoietic development. Using biochemical tools we identified a chromatin-remodeling complex called NuRD that interacts with the GATA1 cofactor FOG1 (Hong et al., 2005). We engineered mice in which the FOG1-NuRD interaction is disrupted by point mutations. In collaboration with Drs. Mortimer Poncz and Wei Tong at CHOP we defined multiple defects in hematopoietic progenitor cells and mature lineages (Miccio et al., 2010, Miccio and Blobel, 2011). Notable findings include lineage-inappropriate gene expression of erythroid-megakaryocyte precursors and cardiac septum defects (Gregory et al., 2010) and the unexpected function of the NuRD complex in both repression and activation of transcription. Hence the FOG1-NuRD complex is required not only to promote erythro-megakaryocytic development but for suppression of lineage-inappropriate genes.
Publications
Hong W, Nakazawa M, Chen Y-Y, Kori R, Rakowski C, Blobel GA (2005) FOG-1 recruits the NuRD repressor complex to mediate transcriptional repression by GATA-1. EMBO J 24:2367-2378, 2005. [PMCID: PMC1173144]Miccio A, Wang Y, Hong W, Gregory GD, Wang H, Choi JK, Shelat S, Tong W, Poncz M, and Blobel GA (2010). NuRD mediates activating and repressive functions of GATA-1 and FOG-1 during blood development. EMBO J. 29:442-456. [PMCID: PMC2824460]
Miccio A, and Blobel GA (2010) The role of the GATA-1/FOG-1/NuRD pathway in the expression of human b-like globin genes. Mol Cell Biol 30:3460-70. [PMCID: PMC2897567]
Gregory GD, Miccio A, Bersenev A, Wang Y, Hong W, Zhang Z, Poncz M, Tong W, and Blobel GA (2010). FOG1 requires NuRD to promote hematopoiesis and maintain lineage fidelity within the megakaryocytic–erythroid compartment. Blood 115:2156-66. [PMCID: PMC2844012]
BET proteins
We have a long standing interest in understanding transcription factors and their posttranslational modifications. We found that the hematopoietic regulators GATA1 and NF-E2 are posttranslationally acetylated (Hung et al. 1999, Hung et al., 2001), and are among the first known non-histone nuclear factors described to undergo this modification. We also identified BET family proteins that bind to GATA1 in an acetylation-dependent manner. Mechanistically, we discovered that acetylation is required not for binding to naked DNA templates in vitro but for chromatin association in vivo (Lamonica et al., 2006). Importantly, we discovered that GATA1 acetylation mediates binding to members of the BET bromodomain family, which in turn are required for gene activation but not repression by GATA1 (Lamonica et al., 2011). We are pursuing studies on BET proteins in part because of their great relevance as drug targets for malignant and benign diseases (Stonestrom et al., 2015).
Publications
Hung H.-L., Lau J., .Kim, A.Y., Weiss M.J., and Blobel G.A. (1999). CREB-binding protein acetylates hematopoietic transcription factor GATA-1 at functionally important sites. Mol. Cell. Biol. 19:3496-3505.
Hung H.-L.,Kim A. Y., Hong W., Rakowski C., and Blobel G.A. (2001). Stimulation of NF-E2 DNA binding activity by CBP-mediated acetylation. J.Biol.Chem. 276:10715-10721.
Lamonica J, Vakoc CR, and Blobel GA (2006). Acetylation of GATA-1 is required for chromatin occupancy. Blood 108(12):3736-3738. [PMCID: PMC1895476]
Lamonica JM, Deng W, Kadauke S, Campbell AE, Gamsjaeger R, Wang H, Cheng Y, Billin A, Hardison RC, Mackay JP, and Blobel GA (2011) The double bromodomain protein Brd3 associates with acetylated GATA-1 to promote its chromatin occupancy at erythroid target genes. PNAS 108:159-68. [PMCID 21536911](Selected by Faculty of 1000)
Stonestrom AJ, Hsu SC, Jahn KS, Huang P, Keller CA, Giardine BM, Kadauke S, Campbell AE, Evans P, Hardison RC, and Blobel GA (2015) Function of BET proteins in GATA1-mediated transcriptional activation. Blood 35:1433.