Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting

Chromatin organization and transcriptional regulation are interrelated processes. A shortcoming of current experimental approaches to these complex events is the lack of methods that can capture the activation process on single promoters. We have recently described a method that combines methyltransferase M.SssI treatment of intact nuclei and bisulfite sequencing allowing the representation of replicas of single promoters in terms of protected and unprotected footprint modules. Here we combine this method with computational analysis to study single molecule dynamics of transcriptional activation in the stress inducible GRP78 promoter. We show that a 350–base pair region upstream of the transcription initiation site is constitutively depleted of nucleosomes, regardless of the induction state of the promoter, providing one of the first examples for such a promoter in mammals. The 350–base pair nucleosome-free region can be dissected into modules, identifying transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress. The interaction of the transcriptional machinery with the GRP78 core promoter is highly organized, represented by six major combinatorial states. We show that the TATA box is frequently occupied in the noninduced state, that stress induction results in sequential loading of the endoplasmic reticulum stress response elements, and that a substantial portion of these elements is no longer occupied following recruitment of factors to the transcription initiation site. Studying the positioning of nucleosomes and transcription factors at the single promoter level provides a powerful tool to gain novel insights into the transcriptional process in eukaryotes

Nucleosomes Containing Methylated DNA Stabilize DNA Methyltransferases 3A/3B and Ensure Faithful Epigenetic Inheritance

How epigenetic information is propagated during somatic cell divisions is still unclear but is absolutely critical for preserving gene expression patterns and cellular identity. Here we show an unanticipated mechanism for inheritance of DNA methylation patterns where the epigenetic mark not only recruits the catalyzing enzyme but also regulates the protein level, i.e. the enzymatic product (5-methylcytosine) determines the level of the methylase, thus forming a novel homeostatic inheritance system. Nucleosomes containing methylated DNA stabilize de novo DNA methyltransferases, DNMT3A/3B, allowing little free DNMT3A/3B enzymes to exist in the nucleus. Stabilization of DNMT3A/3B on nucleosomes in methylated regions further promotes propagation of DNA methylation. However, reduction of cellular DNA methylation levels creating more potential CpG substrates counter-intuitively results in a dramatic decrease of DNMT3A/3B proteins due to diminished nucleosome binding and subsequent degradation of the unstable free proteins. These data show an unexpected self-regulatory inheritance mechanism that not only ensures somatic propagation of methylated states by DNMT1 and DNMT3A/3B enzymes but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes

Studies on chromatin assembly in vitro and in transfected cells

Chromatin structure is known to play an active role in regulation of gene expression. One of the features of chromatin in higher eukaryotic cells is that it consists of extensive, regular nucleosome arrays along the DNA sequence. The nucleosome spacing and the degree of regularity can strongly influence chromatin higher-order structure. However, the mechanisms which direct the physiological nucleosome spacing are not well understood. We have developed the first fully defined in vitro chromatin assembly system and have found that histone H5 (or H1) induces physiological nucleosome spacing and extensive ordering on some of the constructs derived from plasmid pBR327. A continuous approximately 800 bp region of the plasmid was required to nucleate histone H5-induced nucleosome alignment, which could then spread to adjacent chromatin. Supporting this idea, a positioned five-nucleosome array appears to originate in the required region. We have also investigated nucleosome structure in chromatin assembled on DNA transfected into cultured animal cells to obtain insight into the mechanisms by which a physiological nucleosome array is generated in vivo. We have observed that every replicating circular DNA does not assemble into a chromatin structure with a regular nucleosome spacing. Simian virus 40 (SV40) DNA transfected into COS-1 cells revealed a much more regular nucleosome spacing than other plasmids. The results suggest that DNA sequences in the SV40 early region are necessary for the regular nucleosome arrangement observed over the whole SV40 genome (5243 bp). Evidence is also provided that periodic nucleosome positioning signals, in two or more phasing frames with respect to DNA, exist throughout the SV40 early region. In addition, we also investigated chromatin assembled on non-replicating plasmid DNA. The data indicate that chromatin assembled on non-replicating plasmid DNA transiently transfected into cultured cells contains an unusual, altered nucleosome structure, and suggest that it might be associated with the nuclear matrix. These properties closely resemble those found for some active genes

A Novel Mechanism of Increased Infections in Contact Lens Wearers

The authors have identified a molecular mechanism that may be the underlying cause of the increase in corneal damage and infection observed in contact lens wearers. The data highlight the biological and financial potential for better contact lens solutions