Tomas Stopka

The ISWI ATPase Snf2h is required for early mouse development.

Chromatin assembly and remodeling complexes alter histone–DNA interactions by using the energy of ATP hydrolysis catalyzed by nucleosome-dependent ATPase subunits. Several classes of ATP-dependent chromatin remodeling complexes exist, including the ISWI family. ISWI complexes disrupt histone–DNA interactions in vitro by facilitating nucleosome sliding. Snf2h is a widely expressed ISWI ATPase. We investigated the role of the Snf2h gene in mammalian development by generating a null mutation in mice. Snf2h heterozygous mutant mice are born at the expected frequency and appear normal. Snf2h-/- embryos die during the periimplantation stage. Blastocyst outgrowth experiments indicate that loss of Snf2h results in growth arrest and cell death of both the trophectoderm and inner cell mass. To investigate the effect of decreased Snf2h levels in adult cells, we performed antisense inhibition of Snf2h in human hematopoietic progenitors. Reducing Snf2h levels inhibited CD34+ progenitors from undergoing cytokine-induced erythropoiesis in vitro. Our results indicate that Snf2h is required for proliferation of early blastocyst-derived stem cells and adult human hematopoietic progenitors. Cells lacking Snf2h are thus prevented from further embryonic development and differentiation.

Modifiers of epigenetic reprogramming show paternal effects in the mouse

There is increasing evidence that epigenetic information can be inherited across generations in mammals, despite extensive reprogramming both in the gametes and in the early developing embryo. One corollary to this is that disrupting the establishment of epigenetic state in the gametes of a parent, as a result of heterozygosity for mutations in genes involved in reprogramming, could affect the phenotype of offspring that do not inherit the mutant allele. Here we show that such effects do occur following paternal inheritance in the mouse. We detected changes to transcription and chromosome ploidy in adult animals. Paternal effects of this type have not been reported previously in mammals and suggest that the untransmitted genotype of male parents can influence the phenotype of their offspring.

PU.1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure

Transcriptional repression mechanisms are important during differentiation of multipotential hematopoietic progenitors, where they are thought to regulate lineage commitment and to extinguish alternative differentiation programs. PU.1 and GATA‐1 are two critical hematopoietic transcription factors that physically interact and mutually antagonize each others transcriptional activity and ability to promote myeloid and erythroid differentiation, respectively. We find that PU.1 inhibits the erythroid program by binding to GATA‐1 on its target genes and organizing a complex of proteins that creates a repressive chromatin structure containing lysine‐9 methylated H3 histones and heterochromatin protein 1. Although these features are thought to be stable aspects of repressed chromatin, we find that silencing of PU.1 expression leads to removal of the repression complex, loss of the repressive chromatin marks and reactivation of the erythroid program. This process involves incorporation of the replacement histone variant H3.3 into nucleosomes. Repression of one transcription factor bound to DNA by another transcription factor not on the DNA represents a new mechanism for downregulating an alternative gene expression program during lineage commitment of multipotential hematopoietic progenitors.

PU.1 and pRB Interact and Cooperate To Repress GATA-1 and Block Erythroid Differentiation

ABSTRACT PU.1 and GATA-1 are two hematopoietic specific transcription factors that play key roles in development of the myeloid and erythroid lineages, respectively. The two proteins bind to one another and inhibit each others function in transcriptional activation and promotion of their respective differentiation programs. This mutual antagonism may be an important aspect of lineage commitment decisions. PU.1 can also act as an oncoprotein since deregulated expression of PU.1 in erythroid precursors causes erythroleukemias in mice. Studies of cultured mouse erythroleukemia cell lines indicate that one aspect of PU.1 function in erythroleukemogenesis is its ability to block erythroid differentiation by repressing GATA-1 (N. Rekhtman, F. Radparvar, T. Evans, and A. I. Skoultchi, Genes Dev. 13:1398-1411, 1999). We have investigated the mechanism of PU.1-mediated repression of GATA-1. We report here that PU.1 binds to GATA-1 on DNA. We localized the repression activity of PU.1 to a small acidic N-terminal domain that interacts with the C pocket of pRB, a well-known transcriptional corepressor. Repression of GATA-1 by PU.1 requires pRB, and pRB colocalizes with PU.1 and GATA-1 at repressed GATA-1 target genes. PU.1 and pRB also cooperate to block erythroid differentiation. Our results suggest that one of the mechanisms by which PU.1 antagonizes GATA-1 is by binding to it at GATA-1 target genes and tethering to these sites a corepressor that blocks transcriptional activity and thereby erythroid differentiation.

Regulation of αA‐crystallin via Pax6, c‐Maf, CREB and a broad domain of lens‐specific chromatin

Pax6 and c‐Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the αA‐crystallin gene, a marker of lens fiber cell differentiation induced by FGF‐signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF‐signaling with αA‐crystallin synthesis. A DCR1/αA‐crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of αA‐crystallin in lens epithelium and fibers. In contrast, the DCR3/αA/EGFP reporter was expressed only in ‘late’ lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the αA‐crystallin promoter in lens chromatin and demonstrated that high levels of αA‐crystallin expression correlate with increased binding of c‐Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9‐hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the αA‐crystallin locus. Our data demonstrate a novel mechanism of Pax6, c‐Maf and CREB function, through regulation of chromatin‐remodeling enzymes, and suggest a multistage model for the activation of αA‐crystallin during lens differentiation.