Emmanuel Käs

Highly preferential nucleation of histone H1 assembly on Scaffold-associated regions

Scaffold-associated regions (SARs) are A + T-rich DNA regions of several hundred base-pairs that are known to bind specifically to nuclear or metaphase scaffolds. Surprisingly, histone H1 specifically associates with SARs. Under conditions of high co-operativity, at input ratios of H1 to DNA up to 15% (w/w), histone H1 binds preferentially to those DNA molecules harboring a SAR, leaving the non-SAR fragments free. Our experiments identify SARs as cis-acting sequences that nucleate co-operative H1 assembly along the SAR into the flanking non-SAR DNA. Experiments with simple DNA polymers implicate homopolymeric oligo(dA).oligo(dT) tracts in preferential histone H1 assembly. The homopolymer oligo(dA).oligo(dT) is, above a critical length of 130 base-pairs, a highly specific nucleator of H1 assembly. SARs may control the conformation of chromatin domains via a regulated H1 assembly and set up the potential transcriptional repertoire of the cell.

A model for chromatin opening: stimulation of topoisomerase II and restriction enzyme cleavage of chromatin by distamycin.

Histone H1 preferentially and cooperatively binds scaffold‐associated regions (SARs) in vitro via specific interactions with the numerous short A + T‐rich tracts (A‐tracts) contained in these sequences. Selective titration of A‐tracts by the oligopeptide distamycin abolishes this interaction and results in a redistribution of H1. Similarly, treatment of intact cells and isolated nuclei with distamycin specifically enhances cleavage of internucleosomal linkers of SARs by topoisomerase II and restriction enzymes. The increased accessibility of these linkers is thought to result from the unfolding (or opening) of the chromatin fiber and to be due to a reduced occupancy by histone H1. Chromatin extraction and H1 assembly experiments support this view. We discuss a model whereby open, H1‐depleted chromatin regions may be generated by titration of A‐tracts by putative distamycin analogues; this local opening may spread to adjacent regions assuming highly cooperative H1‐H1 interactions in chromatin.

BEAF Regulates Cell-Cycle Genes through the Controlled Deposition of H3K9 Methylation Marks into Its Conserved Dual-Core Binding Sites

Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer–promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. Here, we combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element–associated factor (BEAF) subfamily. We identify a dual-core BEAF binding signature at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core–associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. Our results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle.

Resolving the role of topoisomerase II in chromatin structure and function

The catalytic activities of topoisomerase II are responsible primarily for solving the complex topological problems that arise from cellular processes such as DNA replication, transcription and chromosome segregation; however, topoisomerase II may also play a crucial structural role in the chromosome scaffold. Cell-cycle-regulated phosphorylation might be the key to these diverse functions. Phosphorylation of topoisomerase II might trigger the enzymatic activities essential for mitosis and promote interactions with specialized DNA sequences and other protein components of the eukaryotic chromosome to ensure the proper establishment and maintenance of chromatin architecture and function.

The AT-Hook Protein D1 Is Essential for Drosophila melanogaster Development and Is Implicated in Position-Effect Variegation

ABSTRACT We have analyzed the expression pattern of the D1 gene and the localization of its product, the AT hook-bearing nonhistone chromosomal protein D1, during Drosophila melanogaster development. D1 mRNAs and protein are maternally contributed, and the protein localizes to discrete foci on the chromosomes of early embryos. These foci correspond to 1.672- and 1.688-g/cm3 AT-rich satellite repeats found in the centromeric heterochromatin of the X and Y chromosomes and on chromosomes 3 and 4. D1 mRNA levels subsequently decrease throughout later development, followed by the accumulation of the D1 protein in adult gonads, where two distributions of D1 can be correlated to different states of gene activity. We show that the EP473 mutation, a P-element insertion upstream of D1 coding sequences, affects the expression of the D1 gene and results in an embryonic homozygous lethal phenotype correlated with the depletion of D1 protein during embryogenesis. Remarkably, decreased levels of D1 mRNA and protein in heterozygous flies lead to the suppression of position-effect variegation (PEV) of the white gene in the white-mottled (wm4h) X-chromosome inversion. Our results identify D1 as a DNA-binding protein of known sequence specificity implicated in PEV. D1 is the primary factor that binds the centromeric 1.688-g/cm3 satellite repeats which are likely involved in white-mottled variegation. We propose that the AT-hook D1 protein nucleates heterochromatin assembly by recruiting specialized transcriptional repressors and/or proteins involved in chromosome condensation.

Displacement of D1, HP1 and topoisomerase II from satellite heterochromatin by a specific polyamide

The functions of DNA satellites of centric heterochromatin are difficult to assess with classical molecular biology tools. Using a chemical approach, we demonstrate that synthetic polyamides that specifically target AT‐rich satellite repeats of Drosophila melanogaster can be used to study the function of these sequences. The P9 polyamide, which binds the X‐chromosome 1.688 g/cm3 satellite III (SAT III), displaces the D1 protein. This displacement in turn results in a selective loss of HP1 and topoisomerase II from SAT III, while these proteins remain bound to the adjacent rDNA repeats and to other regions not targeted by P9. Conversely, targeting of (AAGAG)n satellite V repeats by the P31 polyamide results in the displacement of HP1 from these sequences, indicating that HP1 interactions with chromatin are sensitive to DNA‐binding ligands. P9 fed to larvae suppresses the position‐effect variegation phenotype of white‐mottled adult flies. We propose that this effect is due to displacement of the heterochromatin proteins D1, HP1 and topoisomerase II from SAT III, hence resulting in stochastic chromatin opening and desilencing of the nearby white gene.

Analysis of NCp7-dependent Activation of HIV-1 cDNA Integration and its Conservation Among Retroviral Nucleocapsid Proteins

HIV-1 nucleocapsid protein NCp7 is a small basic protein with two zinc fingers, found in the virion core where several hundred molecules coat the genomic RNA. NCp7 has nucleic acid chaperone properties that guide reverse transcriptase (RT) to synthesize the proviral DNA flanked by the long terminal repeats (LTR). In vitro, NCp7 can strongly activate magnesium-dependent LTR-DNA strand transfer by integrase (IN). Here we show that IN activation relies on both the basic residues and the zinc fingers of NCp7. NCp7 lacking the zinc fingers binds DNA but moderately stimulates strand transfer by IN. The NCp7 zinc-finger domain binds DNA poorly and does not efficiently stimulate IN activity. However, the NC zinc-finger domain can complement DNA binding to restore full activation of strand transfer by IN. We propose that the basic residues and the zinc fingers function together to stabilize IN at the LTR ends and promote the formation of a nucleoprotein complex competent for integration. We also show that these properties of HIV-1 NCp7 are remarkably conserved among nucleocapsid proteins of retrotransposon and retrovirus origins.

cDNA sequence and expression pattern of the Drosophila melanogaster PAPS synthetase gene: a new salivary gland marker

PAPS synthetase is a bifunctional enzyme containing both ATP sulfurylase and APS kinase activities required for the biosynthesis of PAPS, the sulfate donor in sulfation reactions. Here we report the sequence of the Drosophila melanogaster PAPS synthetase, the first gene implicated in the sulfation pathway to be described in that organism, and the characterization of its specificity of expression in embryos. Whole-mount in situ hybridization reveals that DmPAPSS is a novel salivary gland marker. At the end of embryogenesis, expression of DmPAPSS is also observed at the entry and exit of the gut and the posterior spiracles. We discuss the possibility that the pattern of expression of the DmPAPSS gene might reflect a major role for sulfation in mucus biosynthesis at the end of Drosophila embryogenesis.

RuvAB-directed branch migration of individual Holliday junctions is impeded by sequence heterology

The Holliday junction, the key intermediate of recombination, is generated by strand exchange resulting in a covalent connection between two recombining DNA molecules. Translocation of a Holliday junction along DNA, or branch migration, progressively exchanges one DNA strand for another and determines the amount of information that is transferred between two recombining partners. In Escherichia coli, the RuvAB protein complex promotes rapid and unidirectional branch migration of Holliday junctions. We have studied translocation of Holliday junctions using a quantitative biochemical system together with a ‘single‐molecule’ branch migration assay. We demonstrate that RuvAB translocates the junctions through identical DNA sequences in a processive manner with a broad distribution of individual branch migration rates. However, when the complex encounters short heterologous sequences, translocation of the Holliday junctions is impeded. We conclude that translocation of the junctions through a sequence heterology occurs with a probability of bypass being determined both by the length of the heterologous region and the lifetime of the stalled RuvAB complex.

Large-Scale Conformational Flexibility Determines the Properties of AAA+ TIP49 ATPases.

The TIP49a and TIP49b proteins belong to the family of AAA+ ATPases and play essential roles in vital processes such as transcription, DNA repair, snoRNP biogenesis, and chromatin remodeling. We report the crystal structure of a TIP49b hexamer and the comparative analysis of large-scale conformational flexibility of TIP49a, TIP49b, and TIP49a/TIP49b complexes using molecular modeling and molecular dynamics simulations in a water environment. Our results establish key principles of domain mobility that affect protein conformation and biochemical properties, including a mechanistic basis for the downregulation of ATPase activity upon protein hexamerization. These approaches, applied to the lik-TIP49b mutant reported to possess enhanced DNA-independent ATPase activity, help explain how a three-amino acid insertion remotely affects the structure and conformational dynamics of the ATP binding and hydrolysis pocket while uncoupling ATP hydrolysis from DNA binding. This might be similar to the effects of conformations adopted by TIP49 heterohexamers.