Global and stage specific patterns of Krüppel-associated-box zinc finger protein gene expression in murine early embryonic cells.

Highly coordinated transcription networks orchestrate the self-renewal of pluripotent stem cell and the earliest steps of mammalian development. KRAB-containing zinc finger proteins represent the largest group of transcription factors encoded by the genomes of higher vertebrates including mice and humans. Together with their putatively universal cofactor KAP1, they have been implicated in events as diverse as the silencing of endogenous retroelements, the maintenance of imprinting and the pluripotent self-renewal of embryonic stem cells, although the genomic targets and specific functions of individual members of this gene family remain largely undefined. Here, we first generated a list of Ensembl-annotated KRAB-containing genes encoding the mouse and human genomes. We then defined the transcription levels of these genes in murine early embryonic cells. We found that the majority of KRAB-ZFP genes are expressed in mouse pluripotent stem cells and other early progenitors. However, we also identified distinctively cell- or stage-specific patterns of expression, some of which are pluripotency-restricted. Finally, we determined that individual KRAB-ZFP genes exhibit highly distinctive modes of expression, even when grouped in genomic clusters, and that these cannot be correlated with the presence of prototypic repressive or activating chromatin marks. These results pave the way to delineating the role of specific KRAB-ZFPs in early embryogenesis

KRAB/KAP1 Regulation of Embryonic Stem Cell Pluripotent Self-renewal

KRAB-ZFPs constitute the largest family of transcription factors (TFs) encoded by the mouse genome and are known to interact with the co-repressor KAP1. KRAB/KAP1-mediated regulation is essential for several development- and ESC- specific functions, in particular regulation of pluripotent self-renewal. We performed an expression analysis in mouse pluripotent ESCs and differentiated cells. A large number of KRAB-ZFPs was expressed in pluripotent cells and many of them were differentially regulated in other cell types, suggesting that a set of KRAB-ZFPs can be involved in ESC-specific functions. We identified a set of candidate genes, including Zfp459, which show a pluripotency-specific expression pattern. Zfp459 depletion in ESCs induced transcriptional perturbation of pluripotency- and differentiation-associated genes, even if their ability to self-renew or to differentiate was not impaired, and its expression was not required for the formation of blastocysts ex vivo. To understand the precise role of Zfp459 in ESCs further experiments for the identification of its targets are still required. We used a lentiviral vector (LV) to over-express in ESCs ZFP57, which had been previously demonstrated to play a role in regulating genomic imprinting, in fusion with HA-tags. ChIP-seq experiments using a HA antibody demonstrated that ZFP57-HA binds all the known and predicted imprinted loci and identified a hexanucleotide consensus in most of the ZFP57 binding sites co-occupied also by KAP1 and its associated factor SetDB1. We also demonstrated that our LV based expression system could be exploited to perform ChIP-seq analyses in ESCs of TFs, such as Zfp459, for which specific antibodies are not available. Finally, when we cultured ESCs in different conditions, we observed that KAP1 recruitment upstream of Nanog, which had been previously observed, was dependent on the MAPK and GSK3 signaling pathways. We hypothesized that this could be due to the presence in these conditions of ESCs expressing heterogeneous Nanog levels. However, when Nanoghigh and Nanoglow cells were sorted, both the populations shown a comparable enrichment for KAP1 binding upstream of Nanog. The precise role for the KAP1-mediated regulation of Nanog in ESCs and its dependence on extrinsic signaling pathways remains unclear, even if we hypothesize that it can depend on the mono-/bi-allelic expression of Nanog in ESCs and during early development

In Embryonic Stem Cells, ZFP57/KAP1 Recognize a Methylated Hexanucleotide to Affect Chromatin and DNA Methylation of Imprinting Control Regions

The maintenance of H3K9 and DNA methylation at imprinting control regions (ICRs) during early embryogenesis is key to the regulation of imprinted genes. Here, we reveal that ZFP57, its cofactor KAP1, and associated effectors bind selectively to the H3K9me3-bearing, DNA-methylated allele of ICRs in ES cells. KAP1 deletion induces a loss of heterochromatin marks at ICRs, whereas deleting ZFP57 or DNMTs leads to ICR DNA demethylation. Accordingly, we find that ZFP57 and KAP1 associated with DNMTs and hemimethylated DNA-binding NP95. Finally, we identify the methylated TGCCGC hexanucleotide as the motif that is recognized by ZFP57 in all ICRs and in several tens of additional loci, several of which are at least ZFP57-dependently methylated in ES cells. These results significantly advance our understanding of imprinting and suggest a general mechanism for the protection of specific loci against the wave of DNA demethylation that affects the mammalian genome during early embryogenesis

Control of mitophagie by microRNAs - A key step of erythropoiesis

Hematopoiesis is orchestrated by a succession of lineage- and stage-specific transcription factors working in concert with chromatin modifiers. Here, we explored the role of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor KAP1 in this process. The hematopoietic-restricted deletion of Kap1 in the mouse resulted in severe hypoproliferative anemia, with Kap1-deleted erythroblasts failing to induce mitophagy-associated genes, hence to eliminate mitochondria. This was due to persistent expression of microRNAs targeting mitophagy transcripts, itself secondary to a lack of repression by stage-specific KRAB-ZFPs. This KRAB/KAP1-microRNA regulatory cascade is evolutionary conserved, as it also controls mitophagy during human erythropoiesis. A multilayered transcription regulatory system is thus unveiled, where protein- and RNA-based repressors are super-imposed in combinatorial fashion to govern the timely triggering of an essential differentiation event

Data from: Identification of ZEB1 as a central component of the adipogenic gene regulatory network

Adipose tissue is a key determinant of whole body metabolism and energy homeostasis. Unraveling the regulatory mechanisms underlying adipogenesis is therefore highly relevant from a biomedical perspective. Our current understanding of fat cell differentiation is centered on the transcriptional cascades driven by the C/EBP protein family and the master regulator PPARγ. To elucidate further components of the adipogenic gene regulatory network, we performed a large-scale transcription factor (TF) screen overexpressing 734 TFs in mouse pre-adipocytes and probed their effect on differentiation. We identified 23 novel pro-adipogenic TFs and characterized the top ranking TF, ZEB1, as being essential for adipogenesis both in vitro and in vivo. Moreover, its expression levels correlate with fat cell differentiation potential in humans. Genomic profiling further revealed that this TF directly targets and controls the expression of most early and late adipogenic regulators, identifying ZEB1 as a central transcriptional component of fat cell differentiation

KAP1 regulates gene networks controlling mouse B-lymphoid cell differentiation and function

Chromatin remodeling is fundamental for B-cell differentiation. In the present study, we explored the role of KAP1, the cofactor of KRAB-ZFP transcriptional repressors, in this process. B-lymphoid-specific Kap1-KO mice displayed reduced numbers of mature B cells, lower steady-state levels of Abs, and accelerated rates of decay of neutralizing Abs after viral immunization. Transcriptome analyses of Kap1-deleted B splenocytes revealed an up-regulation of PTEN, the enzymatic counteractor of PIK3 signaling, and of genes encoding DNA-damage response factors, cell-cycle regulators, and chemokine receptors. ChIP/seq studies established that KAP1 bound at or close to several of these genes and controlled chromatin status at their promoters. Genome wide, KAP1 binding sites lacked active B cell-specific enhancers and were enriched in repressive histone marks, further supporting a role for this molecule in gene silencing in vivo. Likely responsible for tethering KAP1 to at least some of these targets, a discrete subset of KRAB-ZFPs is enriched in B lymphocytes. Our results therefore reveal the role of KRAB/KAP1-mediated epigenetic regulation in B-cell development and homeostasis