PR-Set7/Set8/KMT5A is the sole enzyme known to catalyze monomethylation of histone H4 lysine 20 (H4K20) and is present only in multicellular organisms that compact a large fraction of their DNA. We found that mouse embryos that are homozygous null mutants for the gene PR-Set7 display early embryonic lethality prior to the eight-cell stage. Death was due to the absence of PR-Set7 catalytic activity, since microinjection of the wild type, but not a catalytically inactive version, into two-cell embryos rescued the phenotype. A lack of PR-Set7 activity resulted not only in depletion of H4K20me1 but also in reduced levels of the H4K20me2/3 marks catalyzed by the Suv4-20h1/h2 enzymes, implying that H4K20me1 may be essential for the function of these enzymes to ensure the dimethylated and trimethylated states. Embryonic stem cells that were inducibly deleted for PR-Set7 passed through an initial G 2 /M phase, but the progeny were defective at the subsequent S and G 2 /M phases, exhibiting a delay in their cell cycle, accumulation at G 2 /M, massive DNA damage, and improper mitotic chromosome condensation. Cell cycle analysis after synchronization indicated that the defects were a consequence of decreased H4K20me1 due to the absence of PR-Set7. Most importantly, the lack of H4K20me1 also resulted in defects in chromosome condensation in interphase nuclei. These results demonstrate the critical role of H4K20 monomethylation in mammals in a developmental context.Posttranslational modifications (PTMs) on histones influence intra-and internucleosomal interactions and thereby contribute to the diversity in nucleosome and chromatin structure that impacts distinct genomic processes. Among these modifications, histone methylation had been considered to be relatively stable, but recent studies demonstrated that, similar to the other PTMs, it too is subject to regulation. Many methylating and demethylating enzymes that target different lysine and arginine residues have been identified. These enzymes have distinct specificities with respect to the methylation status (monomethyl, dimethyl, and trimethyl) of each residue. Furthermore, increasing numbers of proteins harboring the motifs that specifically recognize various methylated residues have been identified. These proteins, or effectors, mediate/regulate elaborate chromatin-based processes, such as gene expression, which are dictated by the presence of the PTMs. Thus, the catalysis/removal of PTMs and their recognition by effectors constitute an intricately designed system that is key to genomic integrity and function.One of the residues of histone H4 that can be monomethylated, dimethylated, or trimethylated is lysine 20. Recent comprehensive analysis of H4 modifications with top-down mass spectrometry in human and in Drosophila melanogaster cells revealed that the dimethyl group is deposited on the majority of total H4K20, indicative of the wide distribution of H4K20me2 on chromatin (18, 34). On the other hand, H4K20me1 and H4K20me3 are relatively few in abundance. The study a...

When pluripotent cells are exposed to a uniform culture environment they routinely display heterogeneous gene expression. Aspects of this heterogeneity, such as Nanog expression, are linked to differences in the propensity of individual cells to either self-renew or commit towards differentiation. Recent findings have provided new insight into the underlying causes of this heterogeneity, which we summarise here using Nanog, a key regulator of pluripotency, as a model gene. We discuss the role of transcription factor heterogeneity in facilitating the intrinsically dynamic and stochastic nature of the pluripotency network, which in turn provides a potential benefit to a population of cells that needs to balance cell fate decisions.

The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.Supplemental material is available for this article.Received January 25, 2014; revised version accepted April 16, 2014. Embryonic cells are characterized by a large degree of plasticity, which is the ability to generate different cell types upon differentiation and is necessary to start a full developmental program. After fertilization, the mouse embryo has the transient capacity to generate both embryonic and extraembryonic cell types, a feature that is referred to as totipotency (Tarkowski 1959;Ishiuchi and Torres-Padilla 2013). This is in contrast to pluripotent cells, which contribute to all three germ layers of the embryo proper, but not to extraembryonic lineages, and therefore have a more restricted potential than totipotent cells. In mice, only the zygote and two-cell stage blastomeres are, strictly speaking, totipotent, since they have the ability to develop into a full organism without the need of carrier cells (Tarkowski 1959;Tarkowski and Wroblewska 1967;Kelly et al. 1978;Ishiuchi and Torres-Padilla 2013). As development progresses, pluripotent cells form in the inner cell mass (ICM) of the blastocyst, accompanied by the activation of pluripotency-associated transcription factors like Nanog and Pou5f1/Oct4 (Nichols et al. 1998;Chambers et al. 2003). The first differentiated embryonic tissue, the trophectoderm (TE), appears morphologically distinguishable and surrounds the ICM in the blastocyst. Thus, during the early stages of development, the mouse embryo undergoes dramatic changes in cellular plasticity.Upon fertilization, embryonic chromatin undergoes intense chromatin remodeling. Indeed, this epigenetic reprogramming of the gametes is thought to be essential to gain totipotency (Sado and Ferguson-Smith 2005;Surani et al. 2007;Hemberger et al. 2009). However, the precise conformation of embryonic chromatin and the way it...

Hepatocyte nuclear factor 4alpha (HNF4alpha) is essential for the establishment and maintenance of liver-specific gene expression. The HNF4alpha gene codes for several isoforms whose developmental and physiological relevance has not yet been explored. HNF4alpha1 and HNF4alpha7 originate from different promoters, while alternative splicing in 3' leads to HNF4alpha2 and HNF4alpha8, respectively. HNF4alpha7/alpha8 were abundantly expressed in embryonic liver and fetal-like hepatoma cells. HNF4alpha1/alpha2 transcripts were up-regulated at birth and represented the only isoforms in adult-like hepatoma cells. In line with its expression profile, HNF4alpha7 activated more avidly than HNF4alpha1 reporter plasmids for genes that are expressed early. The expression patterns of both isoforms together with the differences observed in their transcriptional activities provide elements accounting for fine-tuning of the activity of HNF4alpha. The sequential expression of HNF4alpha7/alpha8 and HNF4alpha1/alpha2 during mouse liver development is the only modification in liver-enriched transcription factors thus far recorded, which parallels the transition from the fetal to the adult hepatic phenotype.

Gametogenesis, the process during which germ cells are generated is essential for reproduction. In mammals, maternal mRNA and proteins present in the oocyte are required to ensure the progression of development until the embryo activates its genome after fertilisation. It is well established that the oocyte synthesises these maternal factors during oocyte growth and then undergoes a quiescent transcriptional period that will be resumed only after fertilisation. However, the mechanisms that govern transcriptional regulation and subsequent silencing during oogenesis are not well understood. Here, we have examined the expression and localisation of the TATA-binding protein (TBP) and the related protein TBP2 (also called TRF3, TBP-related factor 3) during oogenesis and in early mouse embryos. We show that TBP is expressed in the oocytes at the beginning of folliculogenesis, but it is undetectable during further stages of oocyte development, and becomes abundant again only after fertilisation. In contrast to TBP, we found that TBP2 is highly expressed in growing oocytes during folliculogenesis, declines upon ovulation, and is almost undetectable after fertilisation by the two-cell stage. The mirroring localisation profile of TBP and TBP2 suggests different roles for the two proteins in establishing specialised programs of gene expression during oocyte development and in early mouse embryos. Analysis of mutant mouse ovaries in which oocyte-specific factors have been knocked-out suggests that TBP2 is a potential candidate for regulating transcriptional control of oogenesis. Moreover, our results obtained with oocytes lacking the oocyte-specific nuclear chaperone nucleoplasmin 2 suggest that TBP2 function may be related to non-condensed chromatin conformation.

To understand the mechanisms governing the regulation of nuclear receptor (NR) function, we compared the parameters of activation and repression of two isoforms of the orphan receptor hepatocyte nuclear factor (HNF) 4␣. HNF4␣7 and HNF4␣1 differ only in their N-terminal domains, and their expression in the liver is regulated developmentally. We show that the N-terminal activation function (AF)-1 of HNF4␣1 possesses significant activity that can be enhanced through interaction with glucocorticoid receptor-interacting protein 1 (GRIP-1) and cAMP response element-binding protein-binding protein (CBP). In striking contrast, HNF4␣7 possesses no measurable AF-1, implying major functional differences between the isoforms. Indeed, although HNF4␣1 and HNF4␣7 are able to interact via AF-2 with GRIP-1, p300, and silencing mediator for retinoid and thyroid receptors (SMRT), only HNF4␣1 interacts in a synergistic fashion with GRIP-1 and p300. Although both isoforms interact physically and functionally with SMRT, the repression of HNF4␣7 is less robust than that of HNF4␣1, which may be caused by an increased ability of the latter to recruit histone deacetylase (HDAC) activity to target promoters. Moreover, association of SMRT with HDACs enhanced recruitment of HNF4␣1 but not of HNF4␣7. These observations suggest that NR isoformspecific association with SMRT could affect activity of the SMRT complex, implying that selection of HDAC partners is a novel point of regulation for NR activity. Possible physiological consequences of the multiple interactions with these coregulators are discussed. Members of the nuclear receptor (NR)1 superfamily regulate a broad array of physiological processes (1). Their effects are elicited by either activation or repression of a set of genes that harbor DNA motifs recognized by NRs. Although mechanisms of activation are well understood today, repression is beginning to be recognized as a key player in homeostasis (2-4).Gene activation is generally associated with relaxed chromatin structure, which is facilitated by acetylation and methylation of histone N-terminal tails through the recruitment of acetyltransferases or methyltransferases (5, 6). Recruitment of these coregulators to target promoters occurs mainly through direct physical interaction with one of the two activation function modules of NRs, the ligand-dependent AF-2 (7). The role of the other AF, AF-1, is less well studied. Nevertheless, increasing evidence suggests that AF-1 is involved in recruitment of coactivators such as Src-1, GRIP-1, and CBP (8 -11). The mechanisms of cooperative action of the two AFs within a single NR are by and large poorly understood, but it has been shown for estrogen receptor ␣ that the transcription intermediary factor TIF2 mediates such cooperation (12).Gene repression is associated with recruitment of histone deacetylases (HDACs) that remove acetyl groups from histones, provoking the recondensation of chromatin (5, 6). Repression is achieved by recruiting corepressors to regulatory regions through direct ...

We have characterized a 700 bp enhancer element around -6 kb relative to the HNF4alpha1 transcription start. This element increases activity and confers glucocorticoid induction to a heterologous as well as the homologous promoters in differentiated hepatoma cells and is transactivated by HNF4alpha1, HNF4alpha7, HNF1alpha and HNF1beta in dedifferentiated hepatoma cells. A 240 bp sub-region conserves basal and hormone-induced enhancer activity. It contains HNF1, HNF4, HNF3 and C/EBP binding sites as shown by DNase I footprinting and electrophoretic mobility shift assays using nuclear extracts and/or recombinant HNF1alpha and HNF4alpha1. Mutation analyses showed that the HNF1 site is essential for HNF1alpha transactivation and is required for full basal enhancer activity, as is the C/EBP site. Glucocorticoid response element consensus sites which overlap the C/EBP, HNF4 and HNF3 sites are crucial for optimal hormonal induction. We present a model that accounts for weak expression of HNF4alpha1 in the embryonic liver and strong expression in the newborn/adult liver via the binding sites identified in the enhancer.

The first events of the development of any embryo are under maternal control until the zygotic genome becomes activated. In the mouse embryo, the major wave of transcription activation occurs at the 2-cell stage, but transcription starts already at the zygote (1-cell) stage. Very little is known about the molecules involved in this process. We show that the transcription intermediary factor 1 α (TIF1α) is involved in modulating gene expression during the first wave of transcription activation. At the onset of genome activation, TIF1α translocates from the cytoplasm into the pronuclei to sites of active transcription. These sites are enriched with the chromatin remodelers BRG-1 and SNF2H. When we ablate TIF1α through either RNA interference (RNAi) or microinjection of specific antibodies into zygotes, most of the embryos arrest their development at the 2–4-cell stage transition. The ablation of TIF1α leads to mislocalization of RNA polymerase II and the chromatin remodelers SNF2H and BRG-1. Using a chromatin immunoprecipitation cloning approach, we identify genes that are regulated by TIF1α in the zygote and find that transcription of these genes is misregulated upon TIF1α ablation. We further show that the expression of some of these genes is dependent on SNF2H and that RNAi for SNF2H compromises development, suggesting that TIF1α mediates activation of gene expression in the zygote via SNF2H. These studies indicate that TIF1α is a factor that modulates the expression of a set of genes during the first wave of genome activation in the mouse embryo.