Drosophila DSP1 and Rat HMGB1 Have Equivalent DNA Binding Properties and Share a Similar Secondary Fold (original) (raw)
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BMC Genomics, 2014
Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. While these proteins are almost certainly important for gene regulation they have been studied far less than the core histone proteins. Here we describe the genomic distributions and functional roles of two chromatin architectural proteins: histone H1 and the high mobility group protein HMGD1 in Drosophila S2 cells. Using ChIP-seq, biochemical and gene specific approaches, we find that HMGD1 binds to highly accessible regulatory chromatin and active promoters. In contrast, H1 is primarily associated with heterochromatic regions marked with repressive histone marks. We find that the ratio of HMGD1 to H1 binding is a better predictor of gene activity than either protein by itself, which suggests that competition between these proteins is important for gene regulation. Using knockdown experiments, we show that HMGD1 and H1 affect the occupancy of the other protein, change nucleosome repeat length and modulate gene expression. Collectively, our data suggest that dynamic and mutually exclusive binding of H1 and HMGD1 to nucleosomes and their linker sequences may control the fluid chromatin structure that is required for transcriptional regulation. This study provides a framework to further study the interplay between chromatin architectural proteins and epigenetics in gene regulation. Author contributions: NN, RM, JM, RM, and YNF-M performed the experiments. NN, and YNF-M performed the experimental analyses. GM, JKP and YNF-M performed the bioinformatics analyses. N.N, GM, RM, JM, JKP and YNF-M wrote the paper. N.N, GM, JKP and YNF-M conceived and designed experiments. YNF-M directed the research. Methods S2 cell culture and siRNA knockdown D. Melanogaster S2-DRSC cells (obtained from the Drosophila Genomics Resource Center) were cultured in Schneider's Drosophila medium (Invitrogen) supplemented with 10% FCS (Hyclone). Knockdowns in S2 cells were done using the following constructs: an H1 construct obtained from (Lu et al. 2009) and HMGD1 PCR products were Agresti A, Bianchi ME. 2003. HMGB proteins and gene expression. Current opinion in genetics & development 13(2): 170-178.
IUBMB Life, 1998
Mice have two structural homologues of the Drosophila HP1 gene, termed M31 and M32, which have two structurally conserved domains, the chromo (C) and chromo shadow (CS) domains. In Drosophila, HP1 has been isolated as one of the components of conserved chromatin structure (heterochromatin) and is thought to bind DNA indirectly by mediating formation of a complex of nuclear proteins. However, little is known about the function of M31 and M32 in mammals. In order to assess the function of the M3 t and M32 proteins, Northern blot analysis was performed in mice. M32 mRNA (with approximately 1,800 nucleotides (ntds)) was expressed strongly throughout embryonic stages examined and in TT2 embryonic stem (ES) cells, while expression of M31 mRNA (with approximately 2,300 and 1,100 ntds) was strong in embryos at embryonic day 8.5 (E8.5) and in TT2 cells, but was decreased at E12.5 and thereafter. In adults, expression of M32 mRNA was strongest in brain and brain stem among tissues examined, and was relatively weak in organs including kidney, stomach, intestine and testis. The two M31 transcripts were weakly but uniformly expressed throughout the organs examined in adults. These findings suggest that M31 and M32 may play different roles in murine embryogenesis, although they have the C and CS domains and are members of the same gene family. detected the presence of a second C domain in the C-terminal region of HP1, and termed this domain chromo shadow (CS) domain. The C-terminal region of HPI containing the CS domain was later shown to be responsible for its targeting to heterochromatin, using deletion mutants in the CS domain (5). To date, many C domain-containing nuclear proteins have been identified for several species and are now categorized as members of the chromo superfamily (6).
Journal of Biological Chemistry
The isolation and sequencing of a cDNA clone coding for the entire sequence of chicken chromosomal protein HMG-14 is described. The open reading frame constitutes only 25% of the transcript; the 5"untranslated region is extremely rich in GC residues; and the 3'untranslated region is highly enriched in AT residues. Comparison with other cDNAs coding for HMG-14 and HMG-17 reveals that the transcripts of genes coding for this family of chromosomal proteins have a characteristic structure. The deduced amino acid sequence is unique and different from all other known HMG-14 and -17 sequences. Analysis of amino acid position identity between the chicken HMG-14 and other HMG-141-17 proteins revealed that the protein has 37% similarity to the HMG-17 group and 69% similarity to the HMG-14 group; therefore, the protein is classified as belonging to the HMG-14 group. Additional analysis leads to the conclusion that the chicken cDNA described here codes for the true homolog of calf and human HMG-14 protein. Comparison of all the known HMG-14 sequences reveals the DNA binding domain is conserved and contains the invariant dodecapeptide PKRRSARLSAKP. The HMG-14 proteins have a distinct charge distribution along the polypeptide chain: while the central region is positively charged the Cterminal domain is negatively charged.
The EMBO …, 1999
The centromeric dodeca-satellite of Drosophila forms altered DNA structures in vitro in which its purinerich strand (G-strand) forms stable fold-back structures, while the complementary C-strand remains unstructured. In this paper, the purification and characterization of DDP1, a single-stranded DNA-binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca-satellite C-strand, is presented. In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca-satellite sequences. DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts. During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes. In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co-localizing with the heterochromatin protein HP1. DDP1 is a multi-KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy. Expression of DDP1 complements a ∆scp160 deletion in yeast. These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin.
Differential Binding of HMG1, HMG2, and a Single HMG Box to Cisplatin-Damaged DNA
Toxicology and Applied Pharmacology, 1996
tion, and chromatin assembly (reviewed by Bustin et al., Differential Binding of HMG1, HMG2, and a Single HMG Box 1990). HMG1 and HMG2 have a close evolutionary relationto Cisplatin-Damaged DNA. FARID, R. S., BIANCHI, M. E., FALCIship and contain two DNA binding domains; these domains OLA, L., ENGELSBERG, B. N., AND BILLINGS, P. C. (1996). Toxicol. are known as HMG boxes Bianchi et Appl. Pharmacol. 141, 532-539. al., 1992). HMG boxes are basic domains of ca. 80 amino The HMG box domain is a DNA binding domain present in the acids which contain three a-helical regions and well-connonhistone chromosomal proteins HMG1 and HMG2 and in other served hydrophobic amino acids within their sequences proteins involved in the regulation of gene expression. Previous Landsman and Bustin, 1993). This studies have demonstrated that HMG1 and HMG2 bind with high DNA binding motif has been identified in a large number affinity to DNA modified with the cancer chemotherapeutic drug of proteins involved in chromatin structure and function and cisplatin (CDDP). In this report, we compare the binding of fullgene expression . Two families of length HMG1 and HMG2 and the HMG boxes present in these HMG box-containing proteins have been reported. The first proteins to that of CDDP-DNA. Complexes between HMG1, family consists of proteins containing two or more HMG HMG2, or HMG Box A / B and CDDP-DNA were stable at §500 boxes, such as the nonhistone chromatin proteins HMG1 mM salt, while complexes between a single HMG box and CDDP-DNA exhibited decreased stability. Analysis of a series of HMG1 and HMG2 and UBF, an RNA polymerase 1 transcription Box A mutant constructs revealed different affinities for CDDPfactor (Jantzen et al., 1990). In the second family are proteins DNA. Two constructs containing a Phe to Ala substitution at containing a single HMG box. Examples of these include position 19 and a Tyr to Gly substitution at position 71, are notetissue-specific transcription factors such as the SRY gene worthy; these peptides exhibited reduced affinity for CDDP-DNA. product (testis-determining factor) (Sinclair et al., 1990) and We have generated a structure of HMG1 Box A and used it, along lymphoid-enhancing factor, LEF1 (Travis et al., 1991). Prowith the results of our binding studies, to model its interaction teins containing two or more HMG boxes bind DNA in a with CDDP-DNA. HMG1 Box A binds in the minor groove of sequence-independent fashion, while LEF1, SRY, and other CDDP-DNA, in agreement with earlier studies. Our model preproteins containing a single HMG box exhibit sequencedicts that Tyr71 partially intercalates and forms an H bond with specific binding (Bianchi et al., 1992; Grosschedl et al., the sugar-phosphate backbone. The model also suggests that Phe 1994; Landsman and Bustin, 1993). A characteristic feature 19 does not directly interact with DNA, and hence an Ala substitution at position 19 may alter protein structure. This model should of all HMG box-containing proteins is their ability to recogprovide a framework for future studies examining HMG Boxnize bent DNA structures such as cruciform and four-way DNA interactions. ᭧
Structure of the HMG box motif in the B-domain of HMG1
The EMBO journal, 1993
The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA-binding domains, each of approximately 80 amino acid residues, and an acidic C-terminal tail. Each folded domain represents an 'HMG box', a sequence motif recently recognized in certain sequence-specific DNA-binding proteins and which also occurs in abundant HMG1-like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA-binding structural motif. We have expressed the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H-NMR spectroscopy. There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues...
H1 and HMGB1: modulators of chromatin structure
Biochemical Society Transactions, 2012
Histone H1 and HMGB1 (high-mobility group protein B1) are the most abundant chromosomal proteins apart from the core histones (on average, one copy per nucleosome and per ten nucleosomes respectively). They are both highly mobile in the cell nucleus, with high on/off rates for binding. In vivo and in vitro evidence shows that both are able to organize chromatin structure, with H1 binding resulting in a more stable structure and HMGB1 binding in a less stable structure. The binding sites for H1 and HMGB1 in chromatin are partially overlapping, and replacement of H1 by HMGB1 through the highly dynamic nature of their binding, possibly facilitated by interaction between them, could result in switching of chromatin states. Binding of HMGB1 to DNA or chromatin is regulated by its long and highly acidic tail, which is also involved in H1 binding. The present article focuses mainly on HMGB1 and its interaction with chromatin and H1, as well as its chaperone role in the binding of certain transcription factors (e.g. p53) to their cognate DNA.
Journal of Biochemistry, 2003
DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ( 253 KKRK 256 ) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.