This proposal investigates epigenetic mechanisms by which transcriptional programs are established and maintained to specify hematopoietic gene expression. Studies in Aim 1 will examine how the interaction between the GATA1-cofactor FOG1 and the NuRD histone deacetylase complex is controlled during hematopoietic gene expression. Using a mouse model in which the FOG1-NuRD interaction is disrupted, we observed that in developing erythroid cells and megakaryocytes NuRD is required for both repression and activation of specific genes. The latter was unexpected in light of the established role of NuRD as co-repressor. Here we will explore mechanisms that discern active from repressive FOG1/NuRD complexes by studying posttranslational regulation of FOG1 and NuRD. The mechanisms that govern distinct activities of the FOG1-NuRD complex are deemed critical for lineage choice and normal cellular maturation. Experiments in Aim 2 explore the mechanisms of epigenetic inheritance of transcriptional programs in hematopoiesis. We will study how specific gene expression patterns are restored after mitosis when transcription is disrupted globally. We will examine the kinetics of disassembly and re- assembly during mitosis of key erythroid transcription factors and their co-factors, such as NuRD, using chromatin immunoprecipitation of synchronized cell populations and live cell imaging. Further, we will study the dynamics of histone modifications at hematopoietic regulatory modules throughout the cell cycle. Mechanistic experiments will explore how mitotic bookmarks are established and interpreted to ensure faithful transmission of transcription patterns throughout the cell cycle.
In Aim 3 we will study the higher order chromatin organization of key hematopoietic genes. Previous work under this grant identified functionally important dynamic long-range chromatin loops between hematopoietic regulatory elements. We will extend these studies by using high- throughput technology called 5C to examine in an unbiased fashion chromatin folding at developmentally controlled genes under distinct conditions and cellular contexts. Moreover, we will explore chromatin loops as potential conveyors of epigenetic information through mitosis.
All Aims benefit from a unique combination of powerful cellular systems and well-characterized gene loci to tackle these fundamental problems in developmental molecular genetics. The role of epigenetic regulation of tissue-specific gene expression is timely as it pertains to mechanisms of lineage fidelity and cellular reprogramming.

Public Health Relevance

The proposed studies are aimed to better understand the mechanisms underlying the formation of the blood lineages and their associated disorders. We examine how nuclear factors and their chemical modifications control the genes that govern the specification and differentiation of blood cells. Moreover, we study mechanisms by which cells "remember" their identity throughout the cell cycle. Progress is this area promises novel approaches to treat blood-related disorders including leukemias and anemias.

Agency
National Institute of Health (NIH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK058044-13
Application #
8702146
Study Section
Hematopoiesis Study Section (HP)
Program Officer
Bishop, Terry Rogers
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Deng, Wulan; Rupon, Jeremy W; Krivega, Ivan et al. (2014) Reactivation of developmentally silenced globin genes by forced chromatin looping. Cell 158:849-60
Campbell, Amy E; Hsiung, Chris C-S; Blobel, Gerd A (2014) Comparative analysis of mitosis-specific antibodies for bulk purification of mitotic populations by fluorescence-activated cell sorting. Biotechniques 56:90-1, 93-4
Deng, Wulan; Blobel, Gerd A (2014) Manipulating nuclear architecture. Curr Opin Genet Dev 25:1-7
Campbell, Amy E; Wilkinson-White, Lorna; Mackay, Joel P et al. (2013) Analysis of disease-causing GATA1 mutations in murine gene complementation systems. Blood 121:5218-27
Hardison, Ross C; Blobel, Gerd A (2013) Genetics. GWAS to therapy by genome edits? Science 342:206-7
Blobel, Gerd A; Hardison, Ross C (2013) A cluster to remember. Cell 154:718-20
Lai, Fan; Orom, Ulf A; Cesaroni, Matteo et al. (2013) Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494:497-501
Lejon, Sara; Thong, Sock Yue; Murthy, Andal et al. (2011) Insights into association of the NuRD complex with FOG-1 from the crystal structure of an RbAp48·FOG-1 complex. J Biol Chem 286:1196-203
Gregory, Gregory D; Miccio, Annarita; Bersenev, Alexey et al. (2010) FOG1 requires NuRD to promote hematopoiesis and maintain lineage fidelity within the megakaryocytic-erythroid compartment. Blood 115:2156-66
Miccio, Annarita; Blobel, Gerd A (2010) Role of the GATA-1/FOG-1/NuRD pathway in the expression of human beta-like globin genes. Mol Cell Biol 30:3460-70

Showing the most recent 10 out of 14 publications