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Jeffrey M Craig

Publications and source records attributed to Jeffrey M Craig.

10 recordsLinked to original sources
PubMed2006

Permissive transcriptional activity at the centromere through pockets of DNA hypomethylation.

DNA methylation is a hallmark of transcriptional silencing, yet transcription has been reported at the centromere. To address this apparent paradox, we employed a fully sequence-defined ectopic human centromere (or neocentromere) to investigate the relationship between DNA methylation and transcription. We used sodium bisulfite PCR and sequencing to determine the methylation status of 2,041 CpG dinucleotides distributed across a 6.76-Mbp chromosomal region containing a neocentromere. These CpG dinucleotides were associated with conventional and nonconventional CpG islands. We found an overall hypermethylation of the neocentric DNA at nonconventional CpG islands that we designated as CpG islets and CpG orphans. The observed hypermethylation was consistent with the presence of a presumed transcriptionally silent chromatin state at the neocentromere. Within this neocentric chromatin, specific sites of active transcription and the centromeric chromatin boundary are defined by DNA hypomethylation. Our data demonstrate, for the first time to our knowledge, a correlation between DNA methylation and centromere formation in mammals, and that transcription and "chromatin-boundary activity" are permissible at the centromere through the selective hypomethylation of pockets of sequences without compromising the overall silent chromatin state and function of the centromere.

Animals↗
PubMed2005

Kiss and break up--a safe passage to anaphase in mitosis and meiosis.

Eukaryotic chromosomes have many challenges to overcome between DNA replication and sister chromatid segregation. If these challenges are not met, cell death or unregulated cell division (cancer) may result. During prophase, chromosomes condense, the nuclear membrane breaks down and cohesins are removed from chromosome arms. In prometaphase, initial spindle attachments are made by sister kinetochores followed by correction of erroneous attachments, centromere oscillation between spindle poles and congression towards the cell's equator. In metaphase, all chromosomes attain stable bipolar spindle attachments and align at the metaphase plate, ready for the metaphase-anaphase transition when all ties between sister chromatids are broken. This review concentrates on recent developments that have revealed the intricacies of these processes. We now know more about how the mechanisms of cohesin removal differ between prophase and the metaphase-anaphase transition, the processes for detection and correction of improper spindle-kinetochore attachments and the concept that tension between sister kinetochores is the driving factor for satisfying the spindle checkpoint. We are also beginning to gain some understanding of the mechanisms behind the co-segregation of sister chromatids at the first meiotic division.

Anaphase↗
PubMed2005

Heterochromatin--many flavours, common themes.

Heterochromatin remains condensed throughout the cell cycle, is generally transcriptionally inert and is built and maintained by groups of factors with each group member sharing a similar function. In mammals, these groups include sequence-specific transcriptional repressors, functional RNA and proteins involved in DNA and histone methylation. Heterochromatin is cemented together via interactions within and between each protein group and is maintained by the cell's replication machinery. It can be constitutive (permanent) or facultative (developmentally regulated) and be any size, from a gene promotor to a whole genome. By studying the formation of facultative heterochromatin, we have gained information about how heterochromatin is assembled. We have discovered that there are many different architectural plans for the building of heterochromatin, leading to a seemingly never-ending variety of heterochromatic loci, with each built according to a general rule.

Animals↗
PubMed2004

Effects of scaffold/matrix alteration on centromeric function and gene expression.

We have previously described a 3.5-Mb domain of enhance scaffold/matrix attachment region (S/MAR) at a human neocentromere, and normal expression of underlying genes within this region. We also reported that partial inhibition of histone deacetylation using 33 nmtrichostatin A (TSA) resulted in a shift in the position of the CENP-A-binding domain within the neocentromere, with no noticeable effects on mitotic segregation function. In this study, 33 nM TSA caused a reduction in the size of the enhanced S/MAR domain of one-half to 1.7 Mb. Treatment with a DNA-intercalating drug distamycin A (DST) at 75 microg/ml resulted in a size reduction of the enhanced S/MAR domain at the neocentromere of two-thirds to 1.2 Mb, and that of the CENP-A-binding domain of 40%, from 330 to 196 kb, with no significant shift in the position of the latter domain. Other DST effects include mitotic chromosomal missegregation, reduction in the levels of Topo IIalpha, CENP-A, CENP-C, and HP1alpha, and an increase in mitotic checkpoint protein BubR1. TSA or DST treatment similarly resulted in a significant reduction, by approximately 20 and 50%, respectively, in the size of the enhanced S/MAR domain at the alpha-satellite DNA of a native chromosome 10 centromere. Transcriptional competence within the neocentromere is overall not noticeably altered by either TSA or DST treatment, as is evident from the absence of any significant increase or decrease in the expression levels of 47 underlying genes tested. These results suggest that a substantial contraction of the S/MAR domain may not be deleterious to centromere function, that disruption of the S/MAR domain directly affects the binding properties of a host of scaffold/matrix and centromeric/pericentric proteins, and that the overall competence and regulation of transcription at the neocentromeric chromatin is similar to those found at the corresponding normal genomic sites.

Animals↗
PubMed2003

Analysis of mammalian proteins involved in chromatin modification reveals new metaphase centromeric proteins and distinct chromosomal distribution patterns.

We have examined the metaphase chromosomal localization of 15 proteins that have previously been described as involved in mammalian chromatin modification and/or transcriptional modulation. Immunofluorescence data indicate that all the proteins localize to human and mouse centromeres, a neocentromere, and the active centromere of a dicentric chromosome, with six of these proteins (Sin3A, PCAF, MYST, MBD2, ORC2, P300/CBP) being demonstrated at mammalian centromeres for the first time. Most of these proteins fall into two distinct chromosomal distribution patterns: (a) kinetochore-associated proteins (Sin3A, PCAF, MYST and BAF180), which colocalize with metaphase kinetochores, but not any of the pericentric and other major heterochromatic regions; and (b) heterochromatin-associated proteins (MeCP2, MBD1, MBD2, ATRX, HP1alpha, HDAC1, HDAC2, DNMT1 and DNMT3b), which colocalize with centromeric/pericentric heterochromatin and all other major heterochromatic sites. A heterogeneous third group (c) consists of the origin recognition complex subunit ORC2 and the histone acetyltransferase P300/CBP, which associate generally with kinetochores in humans and centromeric/pericentric heterochromatin in mouse, with some minor differences in localization. These observations indicate an extensive sharing of protein components involved in chromatin modification at gene loci, centromeres and various chromosomal heterochromatic landmarks. The definition of distinct patterns of chromosomal distribution for these proteins provides a useful basis for the further investigation of the broad-ranging roles of these proteins.

Animals↗
PubMed2003

Centromeric chromatin pliability and memory at a human neocentromere.

We show that Trichostatin A (TSA)-induced partial histone hyperacetylation causes a unidirectional shift in the position of a previously defined binding domain for the centromere-specific histone H3 homologue CENP-A at a human neocentromere. The shift of approximately 320 kb is fully reversible when TSA is removed, but is accompanied by an apparent reduction in the density of CENP-A per unit length of genomic DNA at the neocentromere. TSA treatment also instigates a reversible abolition of a previously defined major domain of differentially delayed replication timing that was originally established at the neocentromeric site. None of these changes has any measurable deleterious effects on mitosis or neocentromere function. The data suggest pliability of centromeric chromatin in response to epigenetic triggers, and the non-essential nature of the regions of delayed replication for centromere function. Reversibility of the CENP-A-binding position and the predominant region of delayed replication timing following removal of TSA suggest strong memory at the original site of neocentromeric chromatin formation.

Acetylation↗
PubMed2003

Transcription within a functional human centromere.

Recent data in yeast and Drosophila suggest a domain-like centromere structure with a modified chromatin core and flanking regions of heterochromatin. We have analyzed a functional human centromere and defined a region of increased chromosome scaffold/matrix attachment that overlaps three other distinct and nonoverlapping domains for constitutive centromere proteins CENP-A and CENP-H, and heterochromatin protein HP1. Transcriptional competency is intact throughout the S/MAR-enriched region and within the CENP-A- and CENP-H-associated chromatin. These results provide insights into the relationship between centromeric chromatin and transcriptional competency in vivo, highlighting the permissibility of transcription within the constitutively modified, nonheterochromatic chromatin of a functional eukaryotic centromere.

Animals↗
PubMed2003

A rapid method of genomic array analysis of scaffold/matrix attachment regions (S/MARs) identifies a 2.5-Mb region of enhanced scaffold/matrix attachment at a human neocentromere.

Human neocentromeres are fully functional centromeres that arise at previously noncentromeric regions of the genome. We have tested a rapid procedure of genomic array analysis of chromosome scaffold/matrix attachment regions (S/MARs), involving the isolation of S/MAR DNA and hybridization of this DNA to a genomic BAC/PAC array. Using this procedure, we have defined a 2.5-Mb domain of S/MAR-enriched chromatin that fully encompasses a previously mapped centromere protein-A (CENP-A)-associated domain at a human neocentromere. We have independently verified this procedure using a previously established fluorescence in situ hybridization method on salt-treated metaphase chromosomes. In silico sequence analysis of the S/MAR-enriched and surrounding regions has revealed no outstanding sequence-related predisposition. This study defines the S/MAR-enriched domain of a higher eukaryotic centromere and provides a method that has broad application for the mapping of S/MAR attachment sites over large genomic regions or throughout a genome.

Cell Line, Transformed↗