Runx1 binds as a dimeric complex to overlapping Runx1 sites within a palindromic element in the human GM-CSF enhancer

Runx1 is a developmentally regulated transcription factor that is essential for haemopoiesis. Runx1 can bind as a monomer to the core consensus sequence TGTGG, but binds more efficiently as a hetero-dimer together with the non-DNA binding protein CBFβ as a complex termed core binding factor (CBF). Here, we demonstrated that CBF can also assemble as a dimeric complex on two overlapping Runx1 sites within the palindromic sequence TGTGGCTGCCCACA in the human granulocyte macrophage colony-stimulating factor enhancer. Furthermore, we demonstrated that binding of Runx1 to the enhancer is rigidly controlled at the level of chromatin accessibility, and is dependent upon prior induction of NFAT and AP-1, which disrupt a positioned nucleosome in this region. We employed in vivo footprinting to demonstrate that, upon activation of the enhancer, both sites are efficiently occupied. In vitro binding assays confirmed that two CBF complexes can bind this site simultaneously, and transfection assays demonstrated that both sites contribute significantly to enhancer function. Computer modelling based on the Runx1/CBFβ/DNA crystal structure further revealed that two molecules of CBF could potentially bind to this class of palindromic sequence as a dimeric complex in a conformation whereby both Runx1 and CBFβ within the two CBF complexes are closely aligned

Assembly of Drosophila Centromeric Chromatin Proteins during Mitosis

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation

Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription

SummaryCentromeres are essential chromosomal structures that mediate accurate chromosome segregation during cell division. Centromeres are specified epigenetically by the heritable incorporation of the centromeric histone H3 variant CENP-A. While many of the primary factors that mediate centromeric deposition of CENP-A are known, the chromatin and DNA requirements of this process have remained elusive. Here, we uncover a role for transcription in Drosophila CENP-A deposition. Using an inducible ectopic centromere system that uncouples CENP-A deposition from endogenous centromere function and cell-cycle progression, we demonstrate that CENP-A assembly by its loading factor, CAL1, requires RNAPII-mediated transcription of the underlying DNA. This transcription depends on the CAL1 binding partner FACT, but not on CENP-A incorporation. Our work establishes RNAPII passage as a key step in chaperone-mediated CENP-A chromatin establishment and propagation

GA4GH: International policies and standards for data sharing across genomic research and healthcare.

The Global Alliance for Genomics and Health (GA4GH) aims to accelerate biomedical advances by enabling the responsible sharing of clinical and genomic data through both harmonized data aggregation and federated approaches. The decreasing cost of genomic sequencing (along with other genome-wide molecular assays) and increasing evidence of its clinical utility will soon drive the generation of sequence data from tens of millions of humans, with increasing levels of diversity. In this perspective, we present the GA4GH strategies for addressing the major challenges of this data revolution. We describe the GA4GH organization, which is fueled by the development efforts of eight Work Streams and informed by the needs of 24 Driver Projects and other key stakeholders. We present the GA4GH suite of secure, interoperable technical standards and policy frameworks and review the current status of standards, their relevance to key domains of research and clinical care, and future plans of GA4GH. Broad international participation in building, adopting, and deploying GA4GH standards and frameworks will catalyze an unprecedented effort in data sharing that will be critical to advancing genomic medicine and ensuring that all populations can access its benefits

A role for the CAL1-partner Modulo in centromere integrity and accurate chromosome segregation in Drosophila.

The relationship between the nucleolus and the centromere, although documented, remains one of the most elusive aspects of centromere assembly and maintenance. Here we identify the nucleolar protein, Modulo, in complex with CAL1, a factor essential for the centromeric deposition of the centromere-specific histone H3 variant, CID, in Drosophila. Notably, CAL1 localizes to both centromeres and the nucleolus. Depletion of Modulo, by RNAi, results in defective recruitment of newly-synthesized CAL1 at the centromere. Furthermore, depletion of Modulo negatively affects levels of CID at the centromere and results in chromosome missegregation. Interestingly, examination of Modulo localization during mitosis reveals it localizes to the chromosome periphery but not the centromere. Combined, the data suggest that rather than a direct regulatory role at the centromere, it is the nucleolar function of modulo which is regulating the assembly of the centromere by directing the localization of CAL1. We propose that a functional link between the nucleolus and centromere assembly exists in Drosophila, which is regulated by Modulo

Modulo RNAi causes mislocalization of GFP-CAL1.

<p>A)Western blot analysis of total extracts from a Modulo RNAi time-course experiment. On day 0 cells were treated with dsRNA. An identical number of cells was taken every 24 h for 8 days and cell extracts were loaded on and SDS-PAGE. Western blots with anti-CAL1 and anti-CID antibodies show no visible changes in CAL1 and CID levels while Modulo became undetectable by day 4. Tubulin is shown as loading control. B) Images of control and Modulo RNAi (RNAi) live cells expressing GFP-CAL1 (green) and mCherry-tubulin (red), shown here as a counterstaining. Note the decrease in the nucleolar GFP-CAL1 upon Modulo RNAi. Bar 10 µm. C) Quantification of the nucleolar GFP-CAL1 signal by scatter dot plot. Dots represent the total GFP-CAL1 signal for individual cells. Black line: average signal, blue error bars: standard error. D) Scatter dot plot of the centromeric GFP-CAL1 signal in control untransfected cells versus Modulo RNAi cells (RNAi). Black line: average signal, blue error bars: standard error.</p

Analysis of Modulo localization in S2 cells and the larval brain.

<p>A) IF with anti-Modulo antibody (red) in S2 cells in interphase and at different mitotic stages (as indicated). Co-staining with anti-CID antibodies (green), shows the lack of significant overlap between the two proteins. DAPI is shown in blue. Bar 5 µm. B) IF with anti-Modulo antibody (red) in larval brain squashes. H3 Ser10p staining indicates mitotic cells. Co-staining with CID (green) confirms the lack of co-localization with Modulo. Bar 20 µm. C) IF with anti-Modulo antibody (red) and anti-CID (green) on mitotic chromosome spreads from S2 cells. DAPI is shown in blue. Bar 1 µm.</p

Modulo is required for proper chromosome segregation.

<p>A) Modulo was depleted in S2 cells by RNAi and chromosome segregation in mitosis was monitored by IF. Staining with Modulo antibody confirmed the successful depletion. Anaphases in cells lacking Modulo (RNAi) displayed lagging and stretched chromosomes at higher frequency than control cells. Bar 5 µm. B) Chromosome segregation was monitored by timelapse microscopy in S2 cells expressing H2B-GFP and mCherry-tubulin. Representative frames for a control and RNAi video are shown. Bar 5 µm. C) Representative anaphases identifiable by H3 Ser10p staining from brain whole-mounts from third instar larvae of <i>mod^lethal8</i> heterozygote mutants (<i>mod/+</i>) and in <i>mod^lethal8</i> homozygote mutants (<i>mod/mod</i>). Frequent stretched and lagging chromosomes were observed in the homozygote mutant (see text for details). Bar 15 µm.</p

Identification of the CAL1 partner, Modulo.

<p>A) Immunofluorescence of S2 cell stably expressing FLAG-CAL1 showing colocalization between FLAG-CAL1 and CID. FLAG is shown in green, CID in blue, Modulo in red and DAPI in gray. Bar 5 µm. B) Immunofluorescence of S2 cell showing co-localization of Modulo (red) and Fibrillarin (nucleolar marker, green). DAPI is shown in gray. Bar 5 µm. C) Western blots of IPs carried out with anti-FLAG beads in untransfected S2 cells (no tag) and cells stably expressing FLAG-CAL1. The top Western blot shows the absence of CAL1 in the no-tag and its presence in the FLAG-CAL1 input and IP. The bottom Western blot shows the presence of Modulo in the input of both no-tag and FLAG-CAL1 and the enrichment of Modulo in the FLAG-CAL1 IP. Modulo runs as a 75 KDa protein while CAL1 runs approximately as a 150 KDa protein. D) Western blots of IPs carried out with beads coupled to anti-Modulo antibodies. Mock indicates control IP where the addition of anti-Modulo antibody was omitted. CAL1 is visible in IPs with the antibody and not in the mock IP.</p