Daniel P. Ryan

Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex.

Nuclear LIM‐only (LMO) and LIM‐homeodomain (LIM‐HD) proteins have important roles in cell fate determination, organ development and oncogenesis. These proteins contain tandemly arrayed LIM domains that bind the LIM interaction domain (LID) of the nuclear adaptor protein LIM domain‐binding protein‐1 (Ldb1). We have determined a high‐resolution X‐ray crystal structure of LMO4, a putative breast oncoprotein, in complex with Ldb1‐LID, providing the first example of a tandem LIM:Ldb1‐LID complex and the first structure of a type‐B LIM domain. The complex possesses a highly modular structure with Ldb1‐LID binding in an extended manner across both LIM domains of LMO4. The interface contains extensive hydrophobic and electrostatic interactions and multiple backbone–backbone hydrogen bonds. A mutagenic screen of Ldb1‐LID, assessed by yeast two‐hybrid and competition ELISA analysis, identified key features at the interface and revealed that the interaction is tolerant to mutation. These combined properties provide a mechanism for the binding of Ldb1 to numerous LMO and LIM‐HD proteins. Furthermore, the modular extended interface may form a general mode of binding to tandem LIM domains.

Histone variants at the transcription start-site

The function of a eukaryotic cell crucially depends on accurate gene transcription to ensure the right genes are expressed whereas unrequired genes are repressed. Therefore, arguably, one of the most important regions in the genome is the transcription start-site (TSS) of protein-coding and non-coding genes. Until recently, understanding the mechanisms that define the location of the TSS and how it is created has largely focused on the role of DNA sequence-specific transcription factors. However, within the nucleus of a eukaryotic cell, transcription occurs in a highly compacted nucleosomal environment, and it is becoming clear that accessibility of the TSS is a key controlling step in transcriptional regulation. It has traditionally been thought that transcription can only proceed once the nucleosomes at the TSS have been evicted. New work suggests otherwise, however, and the focus of this review is to challenge this belief.

The N-terminal Region of Chromodomain Helicase DNA-binding Protein 4 (CHD4) Is Essential for Activity and Contains a High Mobility Group (HMG) Box-like-domain That Can Bind Poly(ADP-ribose).

Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-α-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response.

CHD4 Is a Peripheral Component of the Nucleosome Remodeling and Deacetylase Complex.

Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises ∼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.

A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B

The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron—exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z.

The Chromatin Remodelling Protein CHD1 Contains a Previously Unrecognised C-Terminal Helical Domain.

The packaging of eukaryotic DNA into nucleosomes, and the organisation of these nucleosomes into chromatin, plays a critical role in regulating all DNA-associated processes. Chromodomain helicase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodelling protein that is conserved throughout eukaryotes and has an ability to assemble and organise nucleosomes both in vitro and in vivo. This activity is involved in the regulation of transcription and is implicated in mammalian development and stem cell biology. CHD1 is classically depicted as possessing a pair of tandem chromodomains that directly precede a core catalytic helicase-like domain that is then followed by a SANT-SLIDE DNA-binding domain. Here, we have identified an additional conserved domain C-terminal to the SANT-SLIDE domain and determined its structure by multidimensional heteronuclear NMR spectroscopy. We have termed this domain the CHD1 helical C-terminal (CHCT) domain as it is comprised of five α-helices arranged in a variant helical bundle topology. CHCT has a conserved, positively charged surface and is able to bind DNA and nucleosomes. In addition, we have identified another group of proteins, the as yet uncharacterised C17orf64 proteins, as also containing a conserved CHCT domain. Our data provide new structural insights into the CHD1 enzyme family.

A peptide affinity reagent for isolating an intact and catalytically active multi-protein complex from mammalian cells

We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1-15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST-FOG1(1-45).

1H, 15N and 13C assignments of an intramolecular Lmo2-LIM2/Ldb1-LID complex

Lmo2 is a LIM-only protein involved in hematopoiesis and the development of T-cell acute lymphoblastic leukaemia. Here we report backbone and side chain NMR assignments for an engineered intramolecular complex of the C-terminal LIM domain from Lmo2 tethered to the LIM interaction domain (LID) from LIM domain binding protein 1 (Ldb1).

The interplay between H2A.Z and H3K9 methylation in regulating HP1α binding to linker histone-containing chromatin

Abstract One of the most intensively studied chromatin binding factors is HP1α. HP1α is associated with silenced, heterochromatic regions of the genome and binds to H3K9me3. While H3K9me3 is necessary for HP1α recruitment to heterochromatin, it is becoming apparent that it is not sufficient suggesting that additional factors are involved. One candidate proposed as a potential regulator of HP1α recruitment is the linker histone H1.4. Changes to the underlying make-up of chromatin, such as the incorporation of the histone variant H2A.Z, has also been linked with regulating HP1 binding to chromatin. Here, we rigorously dissected the effects of H1.4, H2A.Z and H3K9me3 on the nucleosome binding activity of HP1α in vitro employing arrays, mononucleosomes and nucleosome core particles. Unexpectedly, histone H1.4 impedes the binding of HP1α but strikingly, this inhibition is partially relieved by the incorporation of both H2A.Z and H3K9me3 but only in the context of arrays or nucleosome core particles. Our data suggests that there are two modes of interaction of HP1α with nucleosomes. The first primary mode is through interactions with linker DNA. However, when linker DNA is missing or occluded by linker histones, HP1α directly interacts with the nucleosome core and this interaction is enhanced by H2A.Z with H3K9me3.