MyD88-dependent dendritic and epithelial cell crosstalk orchestrates immune responses to allergens.

Sensitization to inhaled allergens is dependent on activation of conventional dendritic cells (cDCs) and on the adaptor molecule, MyD88. However, many cell types in the lung express Myd88, and it is unclear how signaling in these different cell types reprograms cDCs and leads to allergic inflammation of the airway. By combining ATAC-seq with RNA profiling, we found that MyD88 signaling in cDCs maintained open chromatin at select loci even at steady state, allowing genes to be rapidly induced during allergic sensitization. A distinct set of genes related to metabolism was indirectly controlled in cDCs through MyD88 signaling in airway epithelial cells (ECs). In mouse models of asthma, Myd88 expression in ECs was critical for eosinophilic inflammation, whereas Myd88 expression in cDCs was required for Th17 cell differentiation and consequent airway neutrophilia. Thus, both cell-intrinsic and cell-extrinsic MyD88 signaling controls gene expression in cDCs and orchestrates immune responses to inhaled allergens.

The human Mi-2/NuRD complex and gene regulation.

The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex is an abundant deacetylase complex with a broad cellular and tissue distribution. It is unique in that it couples histone deacetylation and chromatin remodeling ATPase activities in the same complex. A decade of research has uncovered a number of interesting connections between Mi-2/NuRD and gene regulation. The subunit composition of the enzyme appears to vary with cell type and in response to physiologic signals within a tissue. Here, we review the known subunits of the complex, their connections to signaling networks, and their association with cancer. In addition, we propose a working model that integrates the known biochemical properties of the enzyme with emerging models on how chromatin structure and modification relate to gene activity.

The BCL6-associated transcriptional co-repressor, MTA3, is selectively expressed by germinal centre B cells and lymphomas of putative germinal centre derivation.

Metastasis-associated protein 3 (MTA3) is a recently described cell-type specific component of the Mi-2-NURD transcriptional co-repressor complex that is expressed in breast epithelia and germinal centre B cells. In model B cell lines, MTA3 physically interacts with BCL6 and appears to be instrumental in maintenance of the germinal centre B cell transcriptional programme that precludes premature plasmacytic differentiation. Here, we report selective, in situ cell-type specific expression of MTA3 among lymphoid cells largely confined to the germinal centre B cell compartment. Centroblasts display greater expression than smaller, less proliferative centrocytes, with undetectable expression in quiescent plasma cells. Among B cell neoplasms, germinal centre B cell-like lymphomas likewise exhibit selective expression that generally escalates with increasing proliferative capacity. MTA3 protein expression was, in accord, highly predictive of the germinal centre B cell-like gene expression profile for diffuse large B cell lymphomas. Lastly, relative repression of a subset of known BCL6 targets, including BLIMP1 and p27kip1, was highest in diffuse large B cell lymphomas that co-expressed both MTA3 and BCL6 protein. Together, these novel data suggest a role for MTA3 in BCL6-mediated lymphomagenesis in germinal centre B cell-like neoplasms.

Chromosomal regulation by MeCP2: structural and enzymatic considerations.

The unique properties of eukaryotic DNA modified via methylation of cytosine residues are believed to result from the action of a conserved family of proteins, the MBD family. The prototype member of this family, MeCP2, was isolated independently in two laboratories. One group isolated MeCP2 as a methylated DNA-binding protein, the second as a sequence-specific DNA-binding protein. Multiple lines of evidence suggest that MeCP2 functions in assembly of specialized chromatin architecture. While initial findings pointed to an enzymatic mechanism involving histone modification for transcriptional repression mediated by MeCP2, emerging studies clearly provide exceptions to this model. In a recent study, highly compacted, unique chromatin structures were generated by stoichiometric binding of MeCP2 to model chromatin fibers. These findings support the likelihood that MeCP2 can utilize two independent, but not mutually exclusive, mechanisms to repress transcription: enzymatic and structural mechanisms.

Methyl CpG-binding proteins and transcriptional repression.

Since its discovery, methylation of DNA in mammalian cells has been correlated with transcriptional repression and with specialized chromatin structures. Recently, considerable progress has been reported in the identification of protein factors with a highly conserved DNA interaction surface, termed the methyl CpG-binding domain or MBD. A subset has been biochemically linked to histone deacetylases, suggesting a molecular mechanism for the functional properties of methylated DNA. Despite several obvious attractions, the connection between MBD proteins and histone deacetylases fails to explain all the existing data. In fact, the biochemistry and DNA-binding properties of most MBD family members have not been adequately described and considerable evidence exists for alternative mechanisms in the repression of methylated loci. Null mutations have been generated in mice for several MBD family members, the phenotypes of the mutant animals raise important questions regarding the functions of the MBD family. Here, I review the biochemistry, DNA-binding properties, and genetics of the MBD proteins that are linked to transcriptional repression, namely, MeCP2, MBD1, MBD2, and MBD3. Several models to account for the functional properties of methylated DNA are presented.

A Drosophila MBD family member is a transcriptional corepressor associated with specific genes.

DNA methylation in Drosophila melanogaster is restricted temporally during development and occurs at a significantly lower frequency than in mammals. Thus, the regulatory functions, if any, of this form of DNA modification in Drosophila are unclear. However, the presence of homologs of vertebrate methyl-CpG-binding proteins implies functional consequences for DNA methylation in flies. This work describes the properties of dMBD-like, a Drosophila homolog of vertebrate MBD2 and MBD3. dMBD-like and dMBD-likeDelta (a splice variant) failed to bind model methylated DNA probes, inconsistent with their function as mediators of methyl CpG-directed transcriptional repression. However, the MBD-like proteins exhibit transcriptional and biochemical properties consistent with roles as components of a histone deacetylase-dependent corepressor complex similar to the vertebrate Mi-2 complex. The two proteins are differentially expressed during development, suggesting functional specialization. dMBD-like and/or dMBD-likeDelta is present at the chromocenter on larval polytene chromosomes as well as at discrete bands interspersed along the euchromatic chromosome arms, many of which are coincident with known ecdysone-induced loci. This banding pattern suggests gene-specific regulatory functions for dMBD-like and the Drosophila Mi-2 complex.

Methyl CpG binding proteins: coupling chromatin architecture to gene regulation.

A correlation between DNA methylation and transcriptional silencing has existed for many years. Recently, substantial progress has been reported in the search for proteins that interpret the regulatory information inherent in DNA methylation and translate this information into functional states, resulting in the identification of a family of highly conserved proteins, the MBD family. Direct connections between these proteins and histone modification enzymes have emerged as a common theme, implying that DNA methylation exerts its effects primarily through repressive chromatin architecture. Recent structural determinations of the DNA binding domain of two MBD family members, MeCP2 and MBD1, provide a framework to model the interactions of this family with DNA. Comparative sequence analysis and experimental DNA binding data can be interpreted using this structural framework allowing one to contrast the members of the MBD family with each other and to predict the properties of new family members. The identification of mutations in MeCP2, the founding member of this family, as causal for the neurological developmental disorder Rett Syndrome provides additional information regarding amino acid residues crucial to the functions of this interesting protein family.

Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin.

Cancer cells exhibit a set of unique properties that distinguish them from their normal counterparts. Among these features are increased growth rates, loss of differentiation, escape from cell death pathways, evasion of anti-proliferative signals, a decreased reliance on exogenous growth factors and escape from replicative senescence. Acquisition of these features by malignant cells requires impairment of normal cellular control mechanisms. Over the past few years, it has become increasingly apparent that an important subset of the molecular changes commonly found in cancer cells involves inappropriate regulation of gene expression. This review will address regulatory pathways whose disruption contributes to the malignant phenotype. The failure to deacetylate and thus repress transcription by the Class I histone deacetylases HDAC1 and HDAC2 due to disruption of the Rb family of proteins has been firmly established as a mechanism leading to increases in growth rate and cellular proliferation. Recent data suggest that this regulatory circuit also executes G(1) checkpoint arrest downstream of DNA damage, cellular senescence and contact inhibition. In contrast to this failure to deacetylate, it now seems probable that changes in differentiation status may result in part from inappropriate deacetylation and concomitant transcriptional repression mediated by the Class II histone deacetylases. This inappropriate deacetylation by HDAC4, HDAC5 and HDAC6 follows their relocalization from the cytoplasm to the nucleus. Thus, multiple classical features of cancer cells can be manifested by improper histone deacetylation.

Multiple N-CoR complexes contain distinct histone deacetylases.

N-CoR (nuclear receptor corepressor) is a corepressor for multiple transcription factors including unliganded thyroid hormone receptors (TRs). In vitro, N-CoR can interact with the Sin3 corepressor, which in turn binds to the histone deacetylase Rpd3 (HDAC1), predicting the existence of a corepressor complex containing N-CoR, Sin3, and histone deacetylase. However, previous biochemical studies of endogenous Sin3 complexes have failed to find an N-CoR association. Xenopus laevis eggs and oocytes contain all of the necessary components for transcriptional repression by unliganded TRs. In this study, we report the biochemical fractionation of three novel macromolecular complexes containing N-CoR, two of which possess histone deacetylase activity, from Xenopus egg extract. One complex contains Sin3, Rpd3, and RbAp48; the second complex contains a Sin3-independent histone deacetylase; and the third complex lacks histone deacetylase activity. This study describes the first biochemical isolation of endogenous N-CoR-containing HDAC complexes and illustrates that N-CoR associates with distinct histone deacetylases that are both dependent and independent of Sin3. Immunoprecipitation studies show that N-CoR binds to unliganded TR expressed in the frog oocyte, confirming that N-CoR complexes are involved in repression by unliganded TR. These results suggest that N-CoR targets transcriptional repression of specific promoters through at least two distinct histone deacetylase pathways.

Purification of the MeCP2/histone deacetylase complex from Xenopus laevis.

DNA methylation has long been associated with stable transcriptional silencing and a repressive chromatin structure (reviewed in refs. 1,2). Differential methylation is associated with imprinting, carcinogenesis, silencing of repetitive DNA, and allows for differentiating cells to efficiently shut off unnecessary genes. In vertebrates, where 60-90% of genomic CpG dinucleotides are methylated, methylation-dependent repression is vital for proper embryonic development (3). Microinjection experiments using methylated DNA templates implicate chromatin structure as an underlying mechanism of methylation-dependent silencing (4,5). Methyl-specific transcriptional repression requires chromatin assembly, and can be partially relieved by the histone deacetylase inhibitor Trichostatin A. In addition, several proteins have been identified that specifically bind to methylated DNA (6-8). Two of these methyl-DNA binding proteins, MeCP1 and MeCP2, have been shown to mediate transcriptional repression (6,7). MeCP1 is a relatively uncharacterized complex that requires at least 12 symmetrical methyl-CpGs for DNA binding (6). MeCP2 is a single polypeptide containing a methyl-binding domain capable of binding a single methyl-CpG, and a transcriptional repression domain (9). Recently MeCP2 was shown to interact with the Sin3 corepressor and histone deacetylase (10,11). Changes in the acetylation state of the core histone tails correlates with changes in transcription (reviewed in refs. 12,13), and several transcriptional repression complexes containing histone deacetylases have recently been described (10,14,15).

Active remodeling of somatic nuclei in egg cytoplasm by the nucleosomal ATPase ISWI.

Cloning by the transplantation of somatic nuclei into unfertilized eggs requires a dramatic remodeling of chromosomal architecture. Many proteins are specifically lost from nuclei, and others are taken up from the egg cytoplasm. Recreating this exchange in vitro, we identified the chromatin-remodeling nucleosomal adenosine triphosphatase (ATPase) ISWI as a key molecule in this process. ISWI actively erases the TATA binding protein from association with the nuclear matrix. Defining the biochemistry of global nuclear remodeling may facilitate the efficiency of cloning and other dedifferentiation events that establish new stem cell lineages.

Multiple ISWI ATPase complexes from xenopus laevis. Functional conservation of an ACF/CHRAC homolog.

The nucleosomal ATPase ISWI is the catalytic subunit of several protein complexes that either organize or perturb chromatin structure in vitro. This work reports the cloning and biochemical characterization of a Xenopus ISWI homolog. Surprisingly, whereas we find four complex forms of ISWI in egg extracts, we find no functional homolog of NURF. One of these complexes, xACF, consists of ISWI, Acf1, and a previously uncharacterized protein of 175 kDa. Like both ACF and CHRAC, this complex organizes randomly deposited histones into a regularly spaced array. The remaining three forms include two novel ISWI complexes distinct from known ISWI complexes plus a histone-dependent ATPase complex. This comprehensive biochemical characterization of ISWI underscores the evolutionary conservation of the ACF/CHRAC family.

DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters.

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.

ATP-Dependent histone octamer mobilization and histone deacetylation mediated by the Mi-2 chromatin remodeling complex.

The Mi-2 complex has been implicated in chromatin remodeling and transcriptional repression associated with histone deacetylation. Here, we use a purified Mi-2 complex containing six components, Mi-2, Mta 1-like, p66, RbAp48, RPD3, and MBD3, to investigate the capacity of this complex to destabilize histone-DNA interactions and deacetylate core histones. The Mi-2 complex has ATPase activity that is stimulated by nucleosomes but not by free histones or DNA. This nucleosomal ATPase is relatively inefficient, yet is essential to facilitate both translational movement of histone octamers relative to DNA and the efficient deacetylation of the core histones within a mononucleosome. Surprisingly, ATPase activity had no effect on deacetylation of nucleosomal arrays.

Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes.

ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates. We also perform assays with arrays reconstituted with hyperacetylated or trypsinized histones and isolated histone (H3/H4)(2) tetramers. The results define three groups of the ATP-dependent family of remodeling enzymes. In addition we investigate the ability of an acidic activator to recruit remodeling complexes to nucleosomal arrays. We propose that ATP-dependent chromatin remodeling enzymes share a common reaction mechanism and that a key distinction between complexes is in their mode of regulation or recruitment.

Generation of superhelical torsion by ATP-dependent chromatin remodeling activities.

ATP-dependent chromatin remodeling activities participate in the alteration of chromatin structure during gene regulation. All have DNA- or chromatin-stimulated ATPase activity and many can alter the structure of chromatin; however, the means by which they do this have remained unclear. Here we describe a novel activity for ATP-dependent chromatin remodeling activities, the ability to generate unconstrained negative superhelical torsion in DNA and chromatin. We find that the ability to distort DNA is shared by the yeast SWI/SNF complex, Xenopus Mi-2 complex, recombinant ISWI, and recombinant BRG1, suggesting that the generation of superhelical torsion represents a primary biomechanical activity shared by all Snf2p-related ATPase motors. The generation of superhelical torque provides a potent means by which ATP-dependent chromatin remodeling activities can manipulate chromatin structure.

Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation.

Methylation of DNA at the dinucleotide CpG is essential for mammalian development and is correlated with stable transcriptional silencing. This transcriptional silencing has recently been linked at a molecular level to histone deacetylation through the demonstration of a physical association between histone deacetylases and the methyl CpG-binding protein MeCP2 (refs 4,5). We previously purified a histone deacetylase complex from Xenopus laevis egg extracts that consists of six subunits, including an Rpd3-like deacetylase, the RbA p48/p46 histone-binding protein and the nucleosome-stimulated ATPase Mi-2 (ref. 6). Similar species were subsequently isolated from human cell lines, implying functional conservation across evolution. This complex represents the most abundant form of deacetylase in amphibian eggs and cultured mammalian cells. Here we identify the remaining three subunits of this enzyme complex. One of them binds specifically to methylated DNA in vitro and molecular cloning reveals a similarity to a known methyl CpG-binding protein. Our data substantiate the mechanistic link between DNA methylation, histone deacetylation and transcriptional silencing.

Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte.

We investigated the protein associations and enzymatic requirements for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuclear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear. We cloned Xenopus RbAp48 and SIN3 and show that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase activity and transcriptional repression of the TRbetaA promoter. This repression requires deacetylase activity and nuclear import of RPD3 mediated by a carboxy-terminal nuclear localization signal. Exogenous RPD3 is not incorporated into previously described oocyte deacetylase and ATPase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus. We show that RPD3 associates with RbAp48 through N- and C-terminal contacts and that RbAp48 also interacts with SIN3. Xenopus RbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo. Exogenous RPD3 may be targeted to histones through interaction with endogenous RbAp48 to direct transcriptional repression of the Xenopus TRbetaA promoter in the oocyte nucleus. However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors.