Search

Chromatin is the template for the basic processes of replication and transcription, making the maintenance of chromosomal integrity critical for cell viability. To elucidate how dividing cells respond to alterations in chromatin structure, here we analyse the replication programme of primary cells with altered chromatin configuration caused by the genetic ablation of the HMGB1 gene, or three histone H1 genes. We find that loss of chromatin compaction in H1-depleted cells triggers the accumulation of stalled forks and DNA damage as a consequence of transcription-replication conflicts. In contrast, reductions in nucleosome occupancy due to the lack of HMGB1 cause faster fork progression without impacting the initiation landscape or fork stability. Thus, perturbations in chromatin integrity elicit a range of responses in the dynamics of DNA replication and transcription, with different consequences on replicative stress. These findings have broad implications for our understanding of how defects in chromatin structure contribute to genomic instability.

Because of its very high affinity for DNA, NF-kappaB is believed to make long-lasting contacts with cognate sites and to be essential for the nucleation of very stable enhanceosomes. However, the kinetic properties of NF-kappaB interaction with cognate sites in vivo are unknown. Here, we show that in living cells NF-kappaB is immobilized onto high-affinity binding sites only transiently, and that complete NF-kappaB turnover on active chromatin occurs in less than 30 s. Therefore, promoter-bound NF-kappaB is in dynamic equilibrium with nucleoplasmic dimers; promoter occupancy and transcriptional activity oscillate synchronously with nucleoplasmic NF-kappaB and independently of promoter occupancy by other sequence-specific transcription factors. These data indicate that changes in the nuclear concentration of NF-kappaB directly impact on promoter function and that promoters sample nucleoplasmic levels of NF-kappaB over a timescale of seconds, thus rapidly re-tuning their activity. We propose a revision of the enhanceosome concept in this dynamic framework.

Most nuclear proteins reside on a specific chromatin site only for seconds or less. The hit-and-run model of transcriptional control maintains that transcription complexes are assembled in a stochastic fashion from freely diffusible proteins; this contrasts to models involving stepwise assembly of stable holo complexes. However, the chances of forming a productive complex improve if the binding of one factor promotes the binding of its interactors. We prove here that in living cells, the glucocorticoid receptor and HMGB1 interact only within chromatin and not in the nucleoplasm and decrease each other's mobility. Thus, the formation of a GR-HMGB1-chromatin complex is more likely than one would expect from independent binding to chromatin of GR and HMGB1. Remarkably, this complex is potentially stable, and its disassembly is effected by active, ATP-consuming processes. We propose that kinetic cooperativity among transcription factors in chromatin binding may be a common feature in transcription and DNA transactions.

High mobility group box (HMGB) 1 and 2 are two abundant nonhistone nuclear proteins that have been found in association with chromatin. Previous studies based on immunofluorescence analysis indicated that HMGB1 dissociates from chromosomes during mitosis. In the present work, HMGB1 and 2 subcellular localization was reinvestigated in living cells by using enhanced green fluorescent protein- and Discosome sp. red fluorescent protein-tagged proteins. Contrary to previous reports, HMGB1 and 2 were shown to be present under two forms in mitotic cells, i.e., free and associated with the condensed chromatin, which rapidly exchange. A detailed analysis of HMGB2 interaction with mitotic chromosomes indicated that two sites encompassing HMG-box A and B are responsible for binding. Importantly, this interaction was rapidly inactivated when cells were permeabilized or exposed to chemical fixatives that are widely used in immunodetection techniques. A comparable behavior was also observed for two proteins of the HMG-nucleosome binding (HMGN) group, namely, HMGN1 and HMGN2.

Histones are the protein component of nucleosomes, which are the basic packing unit of chromatin. However, histones are also found in the blood, both as components of nucleosomes leaked out from dead cells, or expelled from neutrophils in the active process of NET formation. Circulating histones contribute to inflammation, and to lethality in sepsis, a hyperinflammatory condition, by interacting with specific receptors, notably toll-like receptor 4 (TLR4). Here, we show that histones are also actively released by LPS-activated macrophages in association with extracellular vesicles. Vesicle-associated histones can be recovered from the plasma of mice with sepsis. Actively released histones are on the outer surface of vesicles and can interact with TLR4. Thus, activated macrophages release histones without dying, at the same time, making their DNA more accessible and communicating to other cells that infection is present. [ABSTRACT FROM AUTHOR]

Core histones package the genome into nucleosomes and control its accessibility to transcription factors. High mobility group proteins (HMGs) are, after histones, the second most abundant chromatin proteins and exert global genomic functions in establishing active or inactive chromatin domains. It is becoming increasingly clear that they also specifically control the expression of a limited number of genes. Moreover, they contribute to the fine tuning of transcription in response to rapid environmental changes. They do so by interacting with nucleosomes, transcription factors, nucleosome-remodelling machines, and with histone H1. [Copyright &y& Elsevier]

High mobility group (HMG) proteins are chromatin proteins endowed with ‘architectural’ capabilities. HMGA proteins are moderately sequence-specific, and help build enhanceosomes by interacting with partner proteins and binding stably to the minor groove of DNA; their acetylation/deacetylation signal enhanceosome assembly or disassembly. HMGBs are much more dynamic proteins: they have no sequence specificity, and help transcription factors and other nuclear proteins bind to their cognate sites by bending the DNA molecule. However, HMGBs are rarely retained within the complex. Similarly, HMGBs interact with nucleosomes and promote their sliding, but remain bound only for fractions of a second. We argue that HMGBs fluidize chromatin — an action that appears opposite to that of histone H1. [Copyright &y& Elsevier]

This article focuses on the high mobility group box (HMGB) chromosomal proteins. The HMGB family comprises the three proteins HMGB1, HMGB2 and HMBG3. The structure of these three proteins is highly conserved, and their biochemical properties are so far indistinguishable. HMGBs are composed of three different domains. The localization of these proteins in most cells is nuclear. In their nuclear identity, HMGB1 and HMGB2 bind to the minor groove of DNA, causing a local distortion of the double helix. They have little or no sequence preference and they are recruited to the site of action by specific DNA binding proteins.