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Aging-related chromatin defects via loss of the NURD complex

Physiological and premature aging are characterized by multiple defects in chromatin structure and accumulation of persistent DNA damage. Here we identify the NURD remodeling complex as a key modulator of these aging-associated chromatin defects. We demonstrate loss of several NURD components during premature and normal aging and we find aging-associated reduction of HDAC1 activity. Silencing of individual NURD subunits recapitulates some chromatin defects associated with aging and we provide evidence that structural chromatin defects precede DNA damage accumulation. These results outline a molecular mechanism for chromatin defects during aging.

structure, loss of methylation at H3K9 and H3K27, down-regulation of the heterochromatin protein HP1, and increased transcription of pericentromeric Satellite III repeats (SatIII) 2, 4. In addition, as normally aged cells, HGPS patient cells exhibit increased steady-state levels of DNA damage 3, 5.
To identify the molecular basis of aging-associated chromatin defects, we performed a yeast two hybrid screen using a C-terminal region of lamin A (aa 562-664) which overlaps with the region deleted in progerin (Fig. 1A). We identified the WD40 domain chromatin protein RBBP4 as a lamin A interactor in four independent experiments. None of the other lamin A fragments tested (aa 1-118, aa 416-568, aa 604-657) interacted with RBBP4 by two-hybrid analysis (data not shown). The interaction between RBBP4 and the aa 562-664 fragment of lamin A was confirmed by GST-pull down ( Supplementary Fig. 1A). RBBP4 and RBBP7 are evolutionary conserved histone binding proteins 10. They are shared subunits of several multi-protein complexes involved in the establishment of heterochromatin including the NUcleosome Remodeling and Deacetylase (NURD) complex 11 and the Polycomb PRC2 complex 12. RBBP4 is also the p48 subunit of the CAF-1 complex, which assembles chromatin upon DNA replication and DNA damage repair 13. In support of a physical interaction between lamin A and RBBP4/7, recombinant forms of both proteins bound in vitro to GST-lamin A (1-664) and to a lesser extend to a C-terminal fragment (residues 390 -646) ( Fig. 1B; Supplementary Fig. 1B). Importantly, neither protein bound to GSTprogerin which lacks residues 607-657 of mature lamin A (Fig. 1B). The in vivo interaction of RBBP4, and more weakly of RBBP7, with lamin A was confirmed by immunoprecipitation of endogenous RBBP4/7 by overexpressed One-Strep (OST)-tagged lamin A (Fig. 1C). These results identify the two histone chaperone proteins RBBP4 and 7 as lamin A interactors.
To ask whether RBBP4 and RBBP7 are involved in aging-associated chromatin defects, we probed their status in HGPS cells. Strikingly, a substantial fraction (48 +/− 2%) of HGPS patient cells showed reduced protein levels of RBBP4 when compared to passage-and agematched controls from healthy donors by quantitative immunofluorescence microscopy as previously described (Fig. 1D, E)2, 3. The protein level of RBBP7 was similarly reduced in HGPS patient cells (Fig. 1D, E). As previously observed for other nuclear defects in HGPS cells, the extent of reduction was variable amongst cells in the population, but its extent was similar to that observed in HGPS for several other nuclear proteins including all isoforms of the heterochromatin protein HP1. Furthermore, loss of RBBP4/7 occurred in the same cells that exhibited lower levels of HP1 pointing to global chromatin defects in those cells (Fig.  1D) 2. Reduction of RBBP4 and RBBP7 in HGPS skin fibroblasts relative to control cells was confirmed by Western blot analysis on total cell lysates ( Fig 1E). Similar observations were made in several independent primary HGPS patient cell lines (data not shown) and, as observed for HP1, the extent of RBBP4/7 reduction increased during cell passaging (data not shown; ref. 3,4). RBBP4 and RBBP7 down-regulation occurred at the protein level since similar levels of mRNAs were present in HGPS and control fibroblasts as determined by RT-PCR (Supplementary Fig. 2A). Consistent with the reduction of RBBP4/7, the levels of the centromeric protein CENP-A was reduced in HGPS cells ( Supplementary Fig. 2B, 2C), in agreement with earlier observations demonstrating loss of CENP-A after concomitant siRNA silencing of RBBP4 and RBBP7 14.
The loss of RBBP4 and RBBP7 was dependent on progerin. Upon controlled induction of a GFP-tagged version of progerin in immortalized wild-type skin fibroblasts using a tetracycline-dependent expression system 6, RBBP4 and RBBP7 were almost completely lost within 4 days when compared with the uninduced control ( Fig 1F). The control protein RAD52 was not affected by GFP-progerin expression (Fig. 1F). Conversely, elimination of progerin pre-mRNA from HGPS skin fibroblasts by a previously characterized RNA morpholino approach2 rescued the levels of RBBP4 when compared to the same cells treated with a scrambled control oligonucleotide ( Supplementary Fig. 2D). In addition, staining of HGPS primary fibroblasts with specific anti-progerin antibodies revealed that expression of progerin inversely correlated with RBBP4 levels in these cells ( Supplementary  Fig. 2E). Collectively, these results demonstrate loss of the histone chaperones RBBP4 and RBBP7 in HGPS cells in a progerin-dependent manner.
Given the role of RBBP4 and RBBP7 in various chromatin remodeling and assembly complexes, we hypothesized that their loss contributes to the changes in chromatin structure and elevated levels of DNA damage that occur during premature and normal aging. To directly test whether the absence of RBBP4/7 is sufficient to cause these defects, we analyzed the status of pericentromeric heterochromatin after siRNA-mediated knockdown of RBBP4 and RBBP7, either alone or in combination (Fig. 2). Efficient knockdown was confirmed by Western blotting (Supplementary Fig. 3A). As expected, cells treated with non-targeting control siRNA oligonucleotides displayed several bright foci of H3K9me3, corresponding to pericentromeric heterochromatin15, as confirmed by partial colocalization of these foci with CREST antibody which recognizes centromeric proteins ( Fig. 2A). Upon simultaneous knock-down of RBBP4 and RBBP7 the focal accumulation of H3K9me3 was lost ( Fig. 2A). Silencing of either RBBP4 or RBBP7 alone was not sufficient to cause heterochromatin alterations ( Fig. 2A), most likely reflecting the functional overlap between these two highly similar proteins. In line with previous observations14 concomitant silencing of RBBP4 and RBBP7 led to loss of the centromeric protein CENP-A ( Supplementary Fig. 3B). To further test whether RBBP4 and RBBP7 are involved in mediating the chromatin defects observed in HGPS cells, we probed the effect of loss of RBBP4 and RBBP7 on DNA SatIII repeat transcription by semi-quantitative RT-PCR (Fig.  2B). SatIII repeats are normally maintained in a transcriptionally silent state but are activated in HGPS cells, most likely as a result of heterochromatin disruption 4, 16. As observed in HGPS cells, simultaneous silencing of RBBP4 and RBBP7 caused a significant increase in SatIII transcription compared to control siRNA treated cells (Fig. 2B).
To test whether loss of RBBP4 and RBBP7 is also sufficient to induce the increased levels of DNA damage present in HGPS cells, we probed RBBP4 and RBBP7 depleted cells for the presence of phosphorylated histone H2AX, a marker of DNA damage 17. Similar to cells from HGPS patients and aged individuals, the percentage of cells containing multiple prominent phospho-H2AX foci increased from 5% in control cells to more than 60% in cells depleted of RBBP4 and RBBP7 (Fig. 2C, D) 3. The increase in endogenous DNA damage upon RBBP4/7 silencing was mirrored by an increased proportion of cells in S-phase as RBBP4 and RBBP7 are shared subunits of multiple protein complexes involved in chromatin metabolism, including the NURD complex and the PRC2 complex 11, 12. In addition, RBBP4 is an integral subunit of the CAF-1 complex 13. To test which of these complexes mediates the aging-associated chromatin defects caused by RBBP4 and RBBP7 loss, we tested the status of other subunits of the NURD, CAF-1 and PRC2 complexes in HGPS cells (Fig. 3). Similar to RBBP4 and RBBP7, two additional subunits of the NURD complex, MTA3 and HDAC1, were largely lost from HGPS cells as judged by quantitative single-cell microscopy and Western blotting ( Fig. 3A-C). Consistent with a possible regulatory role of lamin A on the NURD complex, HDAC1 physically interacted with lamin A in vivo as demonstrated by its immunoprecipitation with OST-lamin A ( Supplementary  Fig. 6A). In contrast, protein levels of p150 and EZH2, the catalytic subunit of the CAF-1 and the PRC2 complexes, respectively, were unaffected in HGPS cells (Fig. 3C). The p60 subunit of the CAF-1 complex was up-regulated in HGPS primary fibroblasts (Fig. 3C), likely as a consequence of persistent DNA damage18. We conclude the NURD complex is lost from HGPS patient cells.
The loss of HDAC1 protein in HGPS cells pointed to the possibility that the observed chromatin defects are caused by loss of cellular HDAC1 deacetylase activity. To test this hypothesis we measured HDAC1 activity in HGPS patient cells. HDAC1 activity in total cell extracts and in HDAC1 immunoprecipitates prepared from HGPS patient cells was reduced by ~ 40% compared to matched control cells (Fig. 3D). To directly probe whether the NURD complex is responsible for the aging-associated chromatin defects, we individually knocked down the NURD subunits HDAC1, MTA3, CHD3 or CHD4 in Hela cells ( Supplementary Fig. 6B). In a manner similar to RBBP4/7 knock down, silencing of any subunit increased the percentage of cells lacking H3K9me3 and HP1γ heterochromatin foci (Fig. 4A, B). Furthermore, shRNA mediated silencing of HDAC1, MTA3, CHD3 or CHD4 in primary human fibroblasts ( Supplementary Fig. 6C, D) increased the percentage of cells containing phospho-H2AX positive foci to similar levels as observed in HGPS cells (Fig. 4C, D). These results demonstrate that HDAC1 activity is reduced in HGPS cells and that loss of NURD complex components is sufficient to recapitulate some aging-associated chromatin defects.
Since chromatin defects associated with HGPS also occur during physiological aging 3, we finally probed the status of the NURD subunits in cells from normally aged individuals. Quantitative immunofluorescence microscopy analysis on cells obtained from two young donors (7 and 9 years, respectively) and passage-matched primary fibroblasts from two old donors (92 and 88 years, respectively), revealed significant differences in RBBP4, RBBP7, and HDAC1 protein levels (Fig 5A-C). Cell populations from old individuals consistently showed a 4-5 fold increase in the percentage of cells with reduced protein levels of RBBP4, RBBP7 and HDAC1 (Fig. 5D). At the single cell level, the extent of reduction of NURD components strictly correlated with each other and with HP1γ levels, albeit less pronounced than that observed for HGPS cells as previously described 3. In contrast to HGPS cells, no loss of CENP-A was detected in normally aged cells. We conclude that loss of the RBBP4, RBBP7, and HDAC1 subunits of NURD is not limited to premature aging but is also a feature of physiologically aged cells.
Here we have identified the NURD chromatin remodeling complex as a mediator of agingassociated chromatin defects. We demonstrate reduction of several NURD subunits in cells from a progeroid syndrome and from normally aged individuals. Loss of any one of these components and reduction of HDAC1 activity is sufficient to recapitulate multiple agingassociated chromatin defects. The in vivo physical interaction of lamin A with RBBP4, RBBP7 and HDAC1 observed here, and previously suggested by biochemical fractionation 19, points towards a regulatory role for the nuclear lamina in the function of the NURD complex in normal cells.
We find that loss of RBBP4/7 is dependent on the presence of progerin and is an early event in the appearance of aging-associated chromatin defects. Upon induction of progerin or upon knock-down of RBBP4 and RBBP7, changes to heterochromatin structure occur and are followed by accumulation of DNA damage. We suggest that loss of critical chromatin components RBBP4/7 is an upstream event in the formation of aging-associated chromatin defects. Loss of RBBP4/7 compromises the establishment and maintenance of histone modifications and higher order chromatin structure, possibly making chromatin more susceptible to DNA damage. The increase in DNA damage might be a consequence of higher susceptibility of the affected chromatin to damage or alternatively due to impaired DNA replication 20. Interestingly, similar to the effects we observe upon silencing of RBBP4 and RBBP7, impairment of the H4K20 histone methyltransferase PR-Set7, which is necessary for proper H4K20 tri-methylation associated with heterochromatin, interferes with DNA replication, arrests cells in S-phase and causes increased levels of DNA damage 21, 22.
Our finding that multiple NURD components are lost in HGPS and normally aged cells suggests that the integrity of the NURD complex is compromised and its components are down-regulated during premature and physiological aging. Whether loss of these proteins occurs by activation of a specific protein degradation program or by interference with the normal homeostasis of the NURD protein complex remains to be determined. The involvement of a specific pathway is suggested by the fact that progerin affects the protein levels of several subunits of the NURD complex, but not of other RBBP4-or RBBP7containing complexes, such as CAF-1 or PRC2. While we cannot rule out that the degradation mechanism of NURD components are distinct in HGPS cells and in normally aged cells, the fact that progerin is also expressed at low levels in cells from normally aged individuals is consistent with a conserved degradation mechanism 3. Overall, these results implicate declining NURD remodeling complex function in aging-associated chromatin defects and accumulation of DNA damage during aging.

Plasmids
Plasmids expressing GST -RBBP4/7 were a kind gift of B. Stillman (Cold Spring Harbor, NY)10. Vectors for in vitro transcription and translation of lamin A and progerin were constructed as follows: an EcoRI/BamHI fragment containing the entire CDS of lamin A (1-664) or progerin was excised from either pEGFP-Lamin A or pEGFP-Lamin Δ50 2 , respectively, and cloned into the pCDNA3.1 (−) vector (Invitrogen) cut with the same enzymes. pGBKT7-lamin A (390-646) was a kind gift of T. Dittmer (NCI/NIH, Maryland). N-terminal One-Strep tag (IBA BioTagnology, Göttingen, Germany) oligonucleotides (Sigma, Zwijndrecht, Netherlands) were cloned into BamHI and EcoRI sites of pBabe puro (Plasmid 1764, Addgene). The retroviral pBabe puro One-Strep-Lamin A plasmid was generated by inserting lamin A from the pBabe puro HA-Lamin A vector23 into the SalI and EcoRI sites. In order to generate a lentiviral expression system a BamHI and EcoRI containing multiple cloning site was introduced in the pCDH1-MCS1-EF1-Puro vector (System BioSciences, Mountainview, California, USA), the puromycin cassette was replaced by a blasticidin cassette from the pLenti6/V5-GW/LacZ vector (Invitrogen, Carlsbad, California, USA) and the One-STrEP-tagged lamin A from the pBabe puro One-Strep-Lamin A vector was ligated into the BamHI and EcoRI restriction sites, thereby generating the lentiviral pCDHblast MCSNard OST-Lamin A vector. The Yeast Two Hybrid screen was performed as described24, 25 siRNA and shRNA constructs siGenome SMART pool siRNA oligos against human RBBP4 and RBBP7 were purchased from Dharmacon-Thermo Scientific and transfected at 0 and 72h into Hela cells at 50 nM using Oligofectamine (Invitrogen) or Dharmafect1 (Dharmacon-Thermo), following the manufacturer instructions. RBBP4:

Immunofluorescence microscopy and image quantitation
Indirect immunofluorescence microscopy was performed as previously described 2.
Quantitative microscopy measurements were performed as previously described 2, 3. For single cell analysis, at least 200 cells per sample were counted in triplicate and the error bars in the histograms represent the standard deviation.

Recombinant proteins expression and in vitro binding assays
GST-fusion proteins were expressed in E. coli BL21(DE3)pLysS (Promega) and affinity purified on glutathione Sepharose beads (GE Healthcare). For in vitro binding assays, GST fusion proteins (1-5 µg) immobilized on glutathione Sepharose beads were prewashed twice in NETN150 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1mM EDTA, 0.5% NP40, 1 mM DTT), incubated 30 min at 4°C with Benzonase, washed once in NETN1000, blocked for 30 min in NETN150, 1 mg/ml BSA and then washed twice in NETN150. [ 35 S]-labelled proteins were generated by in vitro transcription/translation using a T7TNTQuick-rabbit reticulocyte lysate system (Promega). Equal amounts of in vitro translated, [ 35 S]-labelled proteins were incubated in a total volume of 50 µL of NETN150 with either GST or the appropriate GST fusion protein for 1 hr at 4°C in binding buffer. The beads were washed 6× with NETN150, boiled for 5 min in 1× SDS-PAGE Laemmli loading buffer, and resolved on a SDS-PAGE gel. The gel was dried and [ 35 S]-labelled proteins were visualized using a Storm 860 (Molecular Dynamics). All the values were normalized to the internal control Cyclophilin A gene: Cycl-Fw (5′-GTCAACCCCACCGTGTTCTT-3′) and Cycl-Rw (5′-CTGCTGTCTTTGGGACCTTGT -3′).

RNA extraction and RT-PCR
The G-rich strand of SatIII transcripts was retrotranscribed and PCR amplified as previously described 26.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.