The myoD gene converts many differentiated cell types into muscle. MyoD is a member of the basic-helix-loop-helix family of proteins; this 68-amino acid domain in MyoD is necessary and sufficient for myogenesis. MyoD binds cooperatively to muscle-specific enhancers and activates transcription. The helix-loop-helix motif is responsible for dimerization, and, depending on its dimerization partner, MyoD activity can be controlled. MyoD senses and integrates many facets of cell state. MyoD is expressed only in skeletal muscle and its precursors; in nonmuscle cells myoD is repressed by specific genes. MyoD activates its own transcription; this may stabilize commitment to myogenesis.

Injection of Xenopus myoD mRNA into Xenopus embryos leads to only a modest activation of myogenic markers. In contrast, we show that injected mouse myoD mRNA leads to a potent activation. We postulate that XMyoD is under negative control in frog embryos, but because of slight sequence differences, mouse MyoD fails to see the negative signal. Whereas mMyoD is constitutively nuclear, XMyoD is largely cytoplasmic except in a region of the embryo that includes the location where mesoderm induction occurs; there, it is nuclear. At MBT, endogenous XmyoD mRNA is expressed ubiquitously in the frog embryo. Our results suggest that this expression would lead to cytoplasmic XMyoD protein. Among other events, muscle induction might remove this negative regulation, allow MyoD to enter the nucleus, and establish an autoregulatory loop that could commit cells to myogenesis.

ES-cell-based cardiovascular repair requires an in-depth understanding of the molecular mechanisms underlying the differentiation of cardiovascular ES cells. A candidate cardiovascular-fate inducer is the bHLH transcription factor MesP1. As one of the earliest markers, it is expressed specifically in almost all cardiovascular precursors and is required for cardiac morphogenesis. Here we show that MesP1 is a key factor sufficient to induce the formation of ectopic heart tissue in vertebrates and increase cardiovasculogenesis by ES cells. Electrophysiological analysis showed all subtypes of cardiac ES-cell differentiation. MesP1 overexpression and knockdown experiments revealed a prominent function of MesP1 in a gene regulatory cascade, causing Dkk-1-mediated blockade of canonical Wnt-signalling. Independent evidence from ChIP and in vitro DNA-binding studies, expression analysis in wild-type and MesP knockout mice, and reporter assays confirm that Dkk-1 is a direct target of MesP1. Further analysis of the regulatory networks involving MesP1 will be required to preprogramme ES cells towards a cardiovascular fate for cell therapy and cardiovascular tissue engineering. This may also provide a tool to elicit cardiac transdifferentiation in native human adult stem cells.

TGGCA-binding proteins are nuclear proteins with high affinity for double-stranded DNA homologous to the prototype recognition sequence 5'YTGGCANNNTGCCAR 3'. Their ubiquitous tissue distribution in higher vertebrates characterizes them as a class of highly conserved proteins which may exert a basic function. To obtain clues to this function, specific binding sites were mapped on three viral genomes. Recognition sites were identified in the enhancer region of the BK virus, in the LTR of the mouse mammary tumor virus, and in the origin of replication of adenovirus 12. The TGGCA-binding protein from HeLa cells appears to be identical to nuclear factor I described by others, which stimulates initiation of adenovirus DNA replication in vitro. However, data from MMTV, BKV, and from cellular genes suggest that this specific protein-DNA interaction may also be involved in the control of gene activity.

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In Xenopus, cells from the animal hemisphere are competent to form mesodermal tissues from the morula through to the blastula stage. Loss of mesodermal competence at early gastrula is programmed cell-autonomously, and occurs even in single cells at the appropriate stage. To determine the mechanism by which this occurs, we have been investigating a concomitant, global change in expression of H1 linker histone subtypes. H1 histones are usually considered to be general repressors of transcription, but in Xenopus they are increasingly thought to have selective functions in transcriptional regulation. Xenopus eggs and embryos at stages before the midblastula transition are deficient in histone H1 protein, but contain an oocyte-specific variant called histone B4 or H1M. After the midblastula transition, histone B4 is progressively substituted by three somatic histone H1 variants, and replacement is complete by early neurula. Here we report that accumulation of somatic H1 protein is rate limiting for the loss of mesodermal competence. This involves selective transcriptional silencing of regulatory genes required for mesodermal differentiation pathways, like muscle, by somatic, but not maternal, H1 protein.

Chicken TGGCA proteins belong to the ubiquitous, eukaryotic family of NFI-like nuclear proteins, which share an identical DNA binding specificity. They are involved in viral and cellular aspects of transcriptional regulation and they are capable of stimulating Adenovirus initiation of replication. Using microsequencing data from peptides of isolated proteins and PCR supported cloning, we have derived four cDNAs for NFI/TGGCA proteins, which are encoded by three separate chicken genes. Sequence alignments of NFI proteins from chicken and various mammalian species provide evidence for a common genetic equipment among higher eukaryotes, in which several related genes, employing each differential RNA splicing generate an unexpectedly large family of diverse NFI proteins. The extensive similarity of the amino acid sequence throughout the complete coding regions between products of the same gene type in different species indicates a uniform selection pressure on all protein parts, also on those outside the DNA-binding domain.

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.