MeCP2 driven transcriptional repression in vitro: selectivity for methylated DNA, action at a distance and contacts with the basal transcription machinery.

The pathways for selective transcriptional repression of methylated DNA templates by the methyl-CpG-binding protein MeCP2 have been investigated using a purified in vitro transcription system that does not assemble chromatin. MeCP2 selectively inhibits transcription complex assembly on methylated DNA but does not destabilize a pre-assembled transcription complex. MeCP2 functions to repress transcription at a distance of >500 bp from the transcription start site. The transcription repression domain (TRD) of MeCP2 will repress transcription in vitro when fused to a heterologous Gal4 DNA-binding domain. The TRD associates with TFIIB. Exogenous TFIIB does not relieve transcriptional repression established by either intact MeCP2 or a Gal4-TRD fusion protein under these in vitro conditions, nor does the addition of histone deacetylase inhibitors. We find that the transcriptional repression established by both MeCP2 and the Gal4-TRD fusion protein in vitro also correlates with selective assembly of large nucleoprotein complexes. The formation of such complexes reflects a local concentration of DNA-bound transcriptional repressor that may stabilize a state of repression even in the presence of exogenous transcriptional machinery.

A mouse histone H1 variant, H1b, binds preferentially to a regulatory sequence within a mouse H3.2 replication-dependent histone gene.

H1 histones, found in all multicellular eukaryotes, associate with linker DNA between adjacent nucleosomes, presumably to keep the chromatin in a compact, helical state. The identification of multiple histone H1 subtypes in vertebrates suggests these proteins have specialized roles in chromatin organization and thus influence the regulation of gene expression in the multicellular organism. The mechanism by which the association of H1 with nucleosomal DNA is regulated is not completely understood, but affinity for different DNA sequences may play a role. Here we report that a specific H1 subtype in the mouse, namely H1b, selectively binds to a regulatory element within the protein-encoding sequence of a replication-dependent mouse H3.2 gene. We have previously shown that this coding region element, Omega, is the target of very specific interactions in vitro with another nuclear factor called the Omega factor. This element is required for normal gene expression in stably transfected rodent cells. The mouse H1b protein interacts poorly (100-fold lower affinity) with the comparable "Omega" sequence of a replication-independent mouse H3.3 gene. This H3.3 sequence differs at only 4 out of 22 nucleotide positions from the H3.2 sequence. Our findings raise the possibility that this H1b protein plays a specific role in regulation of expression of the replication-dependent histone gene family.

A conserved element in the protein-coding sequence is required for normal expression of replication-dependent histone genes in developing Xenopus embryos.

Replication-dependent histone genes in the mouse and Xenopus share a common regulatory element within the protein-encoding sequence called the CRAS alpha element (coding region activating sequence alpha) which has been shown to mediate normal expression in vivo and to interact with nuclear factors in vitro in a cell cycle-dependent manner. Thus far, the alpha element has only been studied in rodent cells in culture, and its effect on histone gene expression during development has not been determined. Here we examine the role of the alpha element in histone gene expression during Xenopus development which features a switch in histone gene expression from a replication-independent mode in oocytes to a replication-dependent mode in embryos after midblastula stage. In vivo expression experiments involving wild-type or alpha-mutant mouse H3.2 genes show that mutation of the CRAS alpha element results in a fourfold decline of expression in embryos, but does not affect expression in oocytes. Two distinct alpha sequence-specific binding activities were detected in both oocyte and embryonic extracts. A slowly migrating DNA-binding complex was present at relatively constant levels throughout development from the earliest stages of oogenesis through larval stages. In contrast, levels of a rapidly migrating complex were high in stage I and II oocytes, declined in stage II-VI oocytes, remained low in unfertilized eggs and cleavage stage embryos, and rose dramatically after the midblastula transition. The molecular masses of the factors forming the slow and rapidly migrating complexes were estimated to be approximately 110 and 85 kDa, respectively. DNA-binding activity of the 85 kDa alpha-binding factor was affected by phosphorylation, binding with higher affinity in the dephosphorylated state. The abrupt increase in DNA-binding activity of the 85-kDa alpha-binding factor at late blastula coincides with the switch to the replication-dependent mode of histone gene expression. We propose that the conserved alpha element present in the coding sequence of mouse and Xenopus core histone genes is required for normal replication-dependent histone expression in the developing Xenopus embryo.

An H3 coding region regulatory element is common to all four nucleosomal classes of mouse histone-encoding genes.

We have previously identified the alpha element within the mouse H2A and H3 histone gene coding region activating sequences (CRAS). This common element is required for normal in vivo expression of these two replication-dependent genes and interacts with nuclear factor(s). Here we report that the CRAS alpha element is present in the coding region sequences of two other replication-dependent mouse H genes, H2B and H4. The DNA-protein interactions were examined by DNase I footprinting and methylation-interference assays, and are very similar, if not identical, for these replication-dependent genes, confirming that the alpha element is the binding site for common nuclear protein(s) in H genes of all four nucleosomal classes. Moreover, we show that the same nuclear factor is involved in these DNA-protein interactions. Our findings, together with the fact that a replication-independent H gene, H3.3, has a mutated alpha element that fails to interact with nuclear proteins, suggest that this regulatory element is involved in the coordinate expression of the replication-dependent core H genes in the eukaryotic cell cycle.

Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.

Identification of a second conserved element within the coding sequence of a mouse H3 histone gene that interacts with nuclear factors and is necessary for normal expression.

Replication-dependent histone genes of all four nucleosomal classes are coordinately up-regulated at the beginning of S phase of the eukaryotic cell cycle. The universality and importance of this process in eukaryotic cells suggest that common regulatory mechanisms are involved in controlling the high level of expression of these histone genes. We have previously identified the alpha element within mouse H2a.2 and H3.2 coding region activating sequences (CRAS), which is involved in regulation of these two replication-dependent genes. Here we report the identification of a second element within the mouse histone CRAS, the omega element. This element interacts with nuclear proteins and we present in vivo evidence that this sequence is required for normal expression. Omega nucleotides involved in interaction with nuclear proteins have been precisely mapped by menas of DNase I footprinting and methylation interference assays. A naturally occurring mutation in the omega sequence is found in a replication-independent H3.3 gene. Mutation of the H3.2 omega element to that of the H3.3 sequence (3 nt changes) caused a 4-fold drop in in vivo expression of the H3.2 gene in stably transfected CHO cells, equally the effect of mutation of all 7 nt of the element. By UV cross-linking we have determined the approximate molecular weight of the omega binding protein to be 45 kDa. Finally, we identify putative omega sequences in the coding region of mouse H2B and H4 histone genes.