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In the lost group, average nucleosome occupancy increased after treatment and release of CTCF180 , whereas in the gained group nucleosome occupancy at CTCF180 binding sites did not change following CTCF recruitment . Taken together, these findings indicate that CTCF binding and nucleosome occupancy at its binding site are anti-correlated. Post-translational modifications of chromatin proteins are known to play an important role in differential protein binding in chromatin. Poly(ADP-ribosyl)ation is one of such modifications performed by poly(ADP-ribose) polymerases . Phylogenetically ancient PARylation is involved in the regulation of numerous cellular functions, such as DNA repair, replication, transcription, translation, telomere maintenance and chromatin remodeling [16–19]. A growing body of evidence demonstrates the link between CTCF PARylation and its biological functions. For example, the insulator and transcription factor functions of CTCF have been found to be regulated by PARylation .

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Nucleosome occupancy associated with the higher levels of the H3K9me3 is increased in the regions overlapping with CTCF sites in common and lost groups. Molecular changes within regions containing these CTCF sites result in alterations in gene expression patterns. An additional complication of the following analysis is due to a notable ~83% reduction of CTCF associated with the change in the biological states occurred in treated cells . Importantly, the CTCF mRNA levels in treated cells maintained at 59% in comparison with control cells . On the other hand, a reduction of available CTCF would prioritise CTCF binding to stronger DNA sites over weaker sites. CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions.

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A highly PARylated form of CTCF is represented by a protein with an apparent molecular mass 180 kDa , whereas the commonly observed CTCF130, is hypo- or non-PARylated. CTCF130 has been found in many immortalized cell lines and cancer tissues [23, 27–29]. Interestingly, only CTCF180 was detected in normal breast tissues, whereas both CTCF130 and CTCF180 were present in breast tumours . Usually CTCF130 is associated with cell proliferation, whereas CTCF180 is characteristic for non-proliferating cells of different types.

western refining adp

The highly PARylated CTCF form has an apparent molecular mass of 180 kDa , which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130 kDa . The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites).

The latter include cells from healthy breast tissues with very low proliferative index , cells with induced cell cycle arrest, DNA damage , senescence or apoptosis . Currently all existing information regarding the binding characteristics of CTCF has been mined from the experimental data obtained for CTCF130, but not CTCF180. It is not known whether the sets of targets for CTCF130 and CTCF180 are the same, completely different or overlap, and how binding of different forms of CTCF may be associated with alteration in gene expression. One of the reasons for this is that it is difficult to distinguish between CTCF130 and CTCF180 is the absence of an antibody specifically recognising CTCF180. All existing anti-CTCF antibodies detect either only CTCF130 or both CTCF130 and CTCF180.

The GFP-PARP-5a, self-modified with pADPr, was recruited onto magnetic beads coated with anti-GFP IgG during the polymerization reaction, and the beads were collected and washed. As a control, we expressed a GFP fusion of catalytically inactive PARP-5a (GFP-PARP-5a PD) and purified it from lysate on magnetic beads using the same procedure. These beads are coated with PARP-5a polypeptide, but not with pADPr chains. Beads coated with catalytically inactive PARP-5a were unable to assemble microtubule asters in concentrated mitotic lysate . Beads coated with mitotic PARP-5a from which pADPr chains were extended induced robust formation of microtubule asters , suggesting that the presence of pADPr on PARP-5a is required for microtubule aster assembly around the beads. Asters assembled around the PARP-5a-pADPr beads were always located close to beads or bead aggregates, but the microtubules did not seem to emanate directly from the beads. Rather, the asters had distinct foci and appeared to associate laterally with the beads near these foci.

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NuMA was recruited efficiently even at relatively low levels of pADPr modification, unlike another candidate binding protein CH-TOG . This suggests a relatively high-affinity interaction between NuMA and pADPr that is independent of the exact chain length or local density of pADPr. Poly(ADP-ribose) , made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly.

  • Next, we investigated the relationship between the changes in CTCF binding and gene expression.
  • Since PARylation can change physical CTCF interactions with chromatin proteins, we have also looked at the chromatin density profiles based on the Input read density near individual CTCF sites .
  • Examples of specific gene promoter regions where CTCF-associated chromatin rearrangements take place following treatment, together with changes in gene expression patterns, are given in Supplemental Figure S9.
  • We noted that in many cases CTCF binding in control cells was associated with sharp Input peaks in the physical proximity to CTCF, which disappear in treated cells.

The Ran asters are less focused than centrosome-induced asters, as judged by PARP-5a and NuMA fluorescence. Examples of structures that assembled when mitotic chromosomes alone or mitotic chromosomes and centrosomes were incubated in lysate. When chromosomes and centrosomes were mixed, spindle-like assemblies containing dense microtubule bundles were observed. ), so we split it into three fragments for expression and purification . These approximately correspond to the domain organization of NuMA, representing the C- and N-terminal globular domains, and to a central, coiled-coil rod domain. Each segment was expressed in Escherichia coli as a His6 fusion, and purified.

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To determine if the amount of pADPr on the beads affects microtubule aster assembly, we added increasing concentrations of NAD+ to the reaction . Addition of 250 μM and 500 μM NAD+ resulted in a concentration-dependent increase in the number of microtubule asters found associated with the beads, and the microtubule density in each aster . These data show that pADPr elongated from PARP-5a is capable of inducing assembly of pole-like microtubule asters in mitotic cytoplasm, arguing that it has a functional role in pole assembly in cells. We hypothesized that induction of microtubule asters by pADPr-coated beads depended on recruiting specific proteins from the mitotic lysate onto the pADPr polymers and set out to identify these proteins, as describe below. Reconstitution of aspects of spindle pole assembly in concentrated mitotic lysate. To study pADPr contribution to spindle pole assembly, we developed a human cell extract system to recapitulate spindle pole assembly.

western refining adp

The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present. Much of our effort went into developing methods for identifying proteins that bind noncovalently and specifically to pADPr in mitotic cells and might function together with pADPr in spindle pole assembly. We took the approach of incubating PARP-5a modified with pADPr in mitotic lysate and identifying proteins that bound to it. This approach has the advantage that the pADPr is generated by the pole-relevant PARP in mitotic lysate and thus presumably has a physiological pADPr structure.

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Interestingly, when we stratified genes by their expression fold change upon treatment , it appeared that there was a clear preference for retained CTCF at common sites to be associated with genes which did not change or changed their expression minimally. Genes considerably up- or down-regulated upon treatment have lost CTCF . A schematic model illustrating the events observed in control and treated 226LDM cells in which transition from CTCF130 to CTCF180 takes place. Following treatment, cells change morphologically from adherent and flat to suspended and rounded. PARylated CTCF180 in treated cells is largely redistributed from the cell nucleus into cytoplasm . Gained sites characterised by the absence of the CTCF motif acquire CTCF180 after treatment possibly due to interaction with additional proteins or may be just false positives.

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Furthermore, the antibody property differs from batch to batch even for the same commercial vendor, and in order to select the antibody with well-defined properties one has to perform screening of several batches, e.g. using Western blot assays. We next tested if aggregates of pADPr were capable of triggering spindle pole assembly in this system. As a physiological source of spindle pole pADPr, we used magnetic beads coated with mitotic PARP-5a self-modified with pADPr. To make these beads, we prepared dilute mitotic lysate from cells expressing GFP-PARP-5a and supplemented it with 1 mM NAD+ and 1 μM ADP-HPD to promote pADPr synthesis.

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We do not know how the amount of pADPr attached to the PARP-5a in lysate compares to that in spindle poles in living cells. This is difficult to control in lysates, because PARG tends to hydrolyze pADPr, even when a PARG inhibitor was added, so we needed to actively promote PARP activity to observe PARP-5a modification and binding protein recruitment.

western refining adp

A number of studies reported direct interaction between CTCF and poly(ADP-ribose) polymerase 1 , as well as their co-localization in chromatin [23–25]. Furthermore, PARP1 and CTCF have been found to regulate the transition between active and repressed chromatin at the lamina .

In all cases examined, the human cell extracts behaved similar to Xenopus laevis egg extract spindle pole assembly reactions. Concentrated lysates were prepared from mitotic HeLa cells expressing GFP-PARP-5a or GFP-NuMA and supplemented with Alexa 594 tubulin and purified mitotic centrosomes. Microtubules are shown in left panels and Merge, and PARP-5a-GFP or NuMA-GFP in the middle panels, and Merge in the right panels). Note assembly of asters that recruit the known spindle pole proteins PARP-5a and NuMA to their foci. The experiment was performed as in panel a, except that 1 mg/ml RanQ69L was added in place of centrosomes.

We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles. We then performed similar calculations for nucleosome occupancy around common/lost/gained CTCF peaks without refining CTCF sites to CTCF motifs. Figure 5 shows that CTCF130 bound regions in the common control group are associated with smaller average nucleosome occupancy than the same regions in treated cells. This also correlated with the reduced strength of CTCF180 binding at these sites after treatment .

Since PARylation can change physical CTCF interactions with chromatin proteins, we have also looked at the chromatin density profiles based on the Input read density near individual CTCF sites . Examples of specific gene promoter regions where CTCF-associated chromatin rearrangements take place following treatment, together with changes in gene expression patterns, are given in Supplemental Figure S9. We noted that in many cases CTCF binding in control cells was associated with sharp Input peaks in the physical proximity to CTCF, which disappear in treated cells. Next, we investigated the relationship between the changes in CTCF binding and gene expression. By stratifying all genes containing CTCF within +/−10,000 bp from TSS according to their expression level, we observed that it was more likely to find CTCF in the vicinity of a higher expressed gene in both control and treated cells . Furthermore, due to the loss of CTCF near many low-expressed genes upon treatment, this effect is more pronounced in treated cells .