Cheryl A. Keller

An encyclopedia of mouse DNA elements (Mouse ENCODE)

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

The γ2 Subunit of GABAA Receptors Is a Substrate for Palmitoylation by GODZ

The neurotransmitter GABA activates heteropentameric GABAA receptors, which are composed mostly of α, β, and γ2 subunits. Regulated membrane trafficking and subcellular targeting of GABAA receptors is important for determining the efficacy of GABAergic inhibitory function. Of special interest is the γ2 subunit, which is mostly dispensable for assembly and membrane insertion of functional receptors but essential for accumulation of GABAA receptors at synapses. In a search for novel receptor trafficking proteins, we have used the SOS-recruitment system and isolated a Golgi-specific DHHC zinc finger protein (GODZ) as a novel γ2 subunit-interacting protein. GODZ is a member of the superfamily of DHHC cysteine-rich domain (DHHC-CRD) polytopic membrane proteins shown recently in yeast to represent palmitoyltransferases. GODZ mRNA is found in many tissues; however, in brain the protein is detected in neurons only and highly concentrated and asymmetrically distributed in the Golgi complex. GODZ interacts with a cysteine-rich 14-amino acid domain conserved specifically in the large cytoplasmic loop of γ1-3 subunits but not in other GABAA receptor subunits. Coexpression of GODZ and GABAA receptors in heterologous cells results in palmitoylation of the γ2 subunit in a cytoplasmic loop domain-dependent manner. Neuronal GABAA receptors are similarly palmitoylated. Thus, GODZ-mediated palmitoylation represents a novel posttranslational modification that is selective forγ subunit-containing GABAA receptor subtypes, a mechanism that is likely to be important for regulated trafficking of these receptors in the secretory pathway.

GODZ-Mediated Palmitoylation of GABAA Receptors Is Required for Normal Assembly and Function of GABAergic Inhibitory Synapses

Golgi-specific DHHC (Asp-His-His-Cys) zinc finger protein (GODZ) is a DHHC family palmitoyl acyltransferase that is implicated in palmitoylation and regulated trafficking of diverse substrates that function either at inhibitory or excitatory synapses. Of particular interest is the γ2 subunit of GABAA receptors, which is required for targeting these receptors to inhibitory synapses. Here, we report that GODZ and, to a lesser extent, its close paralog sertoli cell gene with a zinc finger domain-β (SERZ-β) are the main members of the DHHC family of enzymes that are able to palmitoylate the γ2 subunit in heterologous cells. Yeast two-hybrid and colocalization assays in human embryonic kidney 293T (HEK293T) cells indicate that GODZ and SERZ-β show indistinguishable palmitoylation-dependent interaction with the γ2 subunit. After coexpression in HEK293T cells, they form homomultimers and heteromultimers, as shown by coimmunoprecipitation and in vivo cross-linking experiments. Analyses in neurons transfected with dominant-negative GODZ (GODZC157S) or plasmid-based GODZ-specific RNAi indicate that GODZ is required for normal accumulation of GABAA receptors at synapses, for normal whole-cell and synaptic GABAergic inhibitory function and, indirectly, for GABAergic innervation. Unexpectedly, GODZ was found to be dispensable for normal postsynaptic AMPA receptor-mediated glutamatergic transmission. We conclude that GODZ-mediated palmitoylation of GABAA receptors and possibly other substrates contributes selectively to the formation and normal function of GABAergic inhibitory synapses.

Principles of regulatory information conservation between mouse and human

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human–mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.

Dynamics of the epigenetic landscape during erythroid differentiation after GATA1 restoration

Interplays among lineage-specific nuclear proteins, chromatin modifying enzymes, and the basal transcription machinery govern cellular differentiation, but their dynamics of action and coordination with transcriptional control are not fully understood. Alterations in chromatin structure appear to establish a permissive state for gene activation at some loci, but they play an integral role in activation at other loci. To determine the predominant roles of chromatin states and factor occupancy in directing gene regulation during differentiation, we mapped chromatin accessibility, histone modifications, and nuclear factor occupancy genome-wide during mouse erythroid differentiation dependent on the master regulatory transcription factor GATA1. Notably, despite extensive changes in gene expression, the chromatin state profiles (proportions of a gene in a chromatin state dominated by activating or repressive histone modifications) and accessibility remain largely unchanged during GATA1-induced erythroid differentiation. In contrast, gene induction and repression are strongly associated with changes in patterns of transcription factor occupancy. Our results indicate that during erythroid differentiation, the broad features of chromatin states are established at the stage of lineage commitment, largely independently of GATA1. These determine permissiveness for expression, with subsequent induction or repression mediated by distinctive combinations of transcription factors.

Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

BackgroundRegulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies.ResultsTAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included.ConclusionsOccupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.

Divergent functions of hematopoietic transcription factors in lineage priming and differentiation during erythro-megakaryopoiesis

Combinatorial actions of relatively few transcription factors control hematopoietic differentiation. To investigate this process in erythro-megakaryopoiesis, we correlated the genome-wide chromatin occupancy signatures of four master hematopoietic transcription factors (GATA1, GATA2, TAL1, and FLI1) and three diagnostic histone modification marks with the gene expression changes that occur during development of primary cultured megakaryocytes (MEG) and primary erythroblasts (ERY) from murine fetal liver hematopoietic stem/progenitor cells. We identified a robust, genome-wide mechanism of MEG-specific lineage priming by a previously described stem/progenitor cell-expressed transcription factor heptad (GATA2, LYL1, TAL1, FLI1, ERG, RUNX1, LMO2) binding to MEG-associated cis-regulatory modules (CRMs) in multipotential progenitors. This is followed by genome-wide GATA factor switching that mediates further induction of MEG-specific genes following lineage commitment. Interaction between GATA and ETS factors appears to be a key determinant of these processes. In contrast, ERY-specific lineage priming is biased toward GATA2-independent mechanisms. In addition to its role in MEG lineage priming, GATA2 plays an extensive role in late megakaryopoiesis as a transcriptional repressor at loci defined by a specific DNA signature. Our findings reveal important new insights into how ERY and MEG lineages arise from a common bipotential progenitor via overlapping and divergent functions of shared hematopoietic transcription factors.

Genome accessibility is widely preserved and locally modulated during mitosis

Mitosis entails global alterations to chromosome structure and nuclear architecture, concomitant with transient silencing of transcription. How cells transmit transcriptional states through mitosis remains incompletely understood. While many nuclear factors dissociate from mitotic chromosomes, the observation that certain nuclear factors and chromatin features remain associated with individual loci during mitosis originated the hypothesis that such mitotically retained molecular signatures could provide transcriptional memory through mitosis. To understand the role of chromatin structure in mitotic memory, we performed the first genome-wide comparison of DNase I sensitivity of chromatin in mitosis and interphase, using a murine erythroblast model. Despite chromosome condensation during mitosis visible by microscopy, the landscape of chromatin accessibility at the macromolecular level is largely unaltered. However, mitotic chromatin accessibility is locally dynamic, with individual loci maintaining none, some, or all of their interphase accessibility. Mitotic reduction in accessibility occurs primarily within narrow, highly DNase hypersensitive sites that frequently coincide with transcription factor binding sites, whereas broader domains of moderate accessibility tend to be more stable. In mitosis, proximal promoters generally maintain their accessibility more strongly, whereas distal regulatory elements tend to lose accessibility. Large domains of DNA hypomethylation mark a subset of promoters that retain accessibility during mitosis and across many cell types in interphase. Erythroid transcription factor GATA1 exerts site-specific changes in interphase accessibility that are most pronounced at distal regulatory elements, but has little influence on mitotic accessibility. We conclude that features of open chromatin are remarkably stable through mitosis, but are modulated at the level of individual genes and regulatory elements.

Functions of BET proteins in erythroid gene expression

Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

2 Drosophila Myogenesis and insights into the Role of nautilus

Several aspects of muscle development appear to be conserved between Drosophila and vertebrate organisms. Among these is the conservation of genes that are critical to the myogenic process, including transcription factors such as nautilus. From a simplistic point of view, Drosophila therefore seems to be a useful organism for the identification of molecules that are essential for myogenesis in both Drosophila and in other species. nautilus, the focal point of this review, appears to be involved in the specification and/or differentiation of a specific subset of muscle founder cells. As with several of its vertebrate and invertebrate counterparts, it is capable of inducing a myogenic program of differentiation reminiscent of that of somatic muscle precursors when expressed in other cell types. We therefore favor the model that nautilus implements the specific differentiation program of these founder cells, rather than their specification. Further analyses are necessary to establish the validity of this working hypothesis. Studies have revealed a critical role for Pax-3 in specifying a particular subset of myogenic cells, the progenitors of the limb muscles. These myogenic cells migrate from the somite into the periphery of the organism, where they differentiate. These myoblasts do not express MyoD or myf5 until they have arrived at their destination and begin the morphologic process of myogenesis (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994). They then begin to express these genes, possibly to put the myogenic plan into action. Thus, as with nautilus, MyoD and myf5 may be necessary for the manifestation of a muscle-specific commitment that has already occurred. By comparison with vertebrates, it was anticipated that the single Drosophila gene would serve the purpose of all four vertebrate genes. However, its restricted pattern of expression and apparent loss-of-function phenotype are inconsistent with this expectation. It remains to be determined whether nautilus functions in a manner similar to just one of the vertebrate genes. Since the myf5- and MyoD-expressing myoblasts are proliferative, the loss of one cell type appears to be compensated by proliferation of the remaining cell type. This apparent plasticity may obscure differences in mutant phenotype resulting from the loss of particular cells that express each of these genes. In Drosophila, by comparison, nautilus-expressing cells committed to the myogenic program undergo few, if any, additional cell divisions, and thus no other cells are available to compensate for the loss of nautilus. Therefore, the apparent differences between the Drosophila nautilus gene and its vertebrate counterparts may reflect, at least in part, differences in the developmental systems rather than differences in the function of the genes themselves.