No abstract

Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.

Eukaryotic DNA replicates asynchronously, with discrete genomic loci replicating during different stages of S phase. Drosophila larval tissues undergo endoreplication without cell division, and the latest replicating regions occasionally fail to complete endoreplication, resulting in underreplicated domains of polytene chromosomes. Here we show that linker histone H1 is required for the underreplication (UR) phenomenon in Drosophila salivary glands. H1 directly interacts with the Suppressor of UR (SUUR) protein and is required for SUUR binding to chromatin in vivo. These observations implicate H1 as a critical factor in the formation of underreplicated regions and an upstream effector of SUUR. We also demonstrate that the localization of H1 in chromatin changes profoundly during the endocycle. At the onset of endocycle S (endo-S) phase, H1 is heavily and specifically loaded into late replicating genomic regions and is then redistributed during the course of endoreplication. Our data suggest that cell cycledependent chromosome occupancy of H1 is governed by several independent processes. In addition to the ubiquitous replication-related disassembly and reassembly of chromatin, H1 is deposited into chromatin through a novel pathway that is replication-independent, rapid, and locus-specific. This cell cycle-directed dynamic localization of H1 in chromatin may play an important role in the regulation of DNA replication timing.

Pu.1 is an ETS family transcription factor (TF) that plays critical roles in erythroid progenitors by promoting proliferation and blocking terminal differentiation. However, the mechanisms controlling expression and down-regulation of Pu.1 during early erythropoiesis have not been defined. In this study, we identify the actions of Runx1 and Pu.1 itself at the Pu.1 gene Upstream Regulatory Element (URE) as major regulators of Pu.1 expression in Burst-Forming Unit erythrocytes (BFUe). During early erythropoiesis, Runx1 and Pu.1 levels decline, and chromatin accessibility at the URE is lost. Ectopic expression of Runx1 or Pu.1, both of which bind the URE, prevents Pu.1 down-regulation and blocks terminal erythroid differentiation, resulting in extensive ex vivo proliferation and immortalization of erythroid progenitors. Ectopic expression of Runx1 in BFUe lacking a URE fails to block terminal erythroid differentiation. Thus, Runx1, acting at the URE, and Pu.1 itself directly regulate Pu.1 levels in erythroid cells, and loss of both factors is critical for Pu.1 down-regulation during terminal differentiation. The molecular mechanism of URE inactivation in erythroid cells through loss of TF binding represents a distinct pattern of Pu.1 regulation from those described in other hematopoietic cell types such as T cells which down-regulate Pu.1 through active repression. The importance of down-regulation of Runx1 and Pu.1 in erythropoiesis is further supported by genome-wide analyses showing that their DNA-binding motifs are highly overrepresented in regions that lose chromatin accessibility during early erythroid development.