Nuclease Sensitivity of Permeabilized Cells Confirms Altered Chromatin Formation at the Fragile X Locus

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1 Somatic Cell and Molecular Genetics, Vol. 22, No.6, 1996, pp Nuclease Sensitivity of Permeabilized Cells Confirms Altered Chromatin Formation at the Fragile X Locus Derek E. Eberhart and Stephen T. Warren Howard Hughes Medical nstitute and Departments of Biochemistry and Pediatrics. Emory University School of Medicine. Atlanta. Georgia 3322 Received 17 July 1996-Final15 October 1996 Abstract-Fragile X syndrome is caused by the expansion and concomitant methylation of a CGG repeat in the 5' untranslated region of the FMR gene which results in the transcriptional silencing of the FMR gene, delayed replication of the FMRl locus, and the formation of a folate sensitive fragile site (FRAXA) at Xq27.3. The mechanism by which repeat expansion and methylation causes these changes is unknown. An in vivo system in which cells were permeabilized with lysophosphatidylcholine followed by digestion with Msp endonuclease was utilized to assess the chromatin conformation at the fragile X locus. The FMR gene was inaccessible to Msp digestion in fragile X patients, but not in normal or carrier individuals, confirming that altered chromatin conformation results from the repeat expansion and methylation seen in fragile X syndrome. NTRODUCTON Fragile X syndrome is a common form of inherited mental retardation caused by the functional absence of FMRP, the protein product of the FMR gene (, 2). FMRP is a ribosomeassociated (3-5), RNA-binding protein (6, 7) with a proposed function in the nuclear export and/or translational control of specific mrnas (5). Nearly all cases of fragile X syndrome are caused by the massive expansion of a COG repeat located in the 5' untranslated region of the FMR gene which leads to aberrant methylation resulting in transcriptional silencing of the gene (8-13). The FMR CGO repeat is polymorphic in length and content with normal individuals having 6-54 repeats (mode 3), with -3 interspersed AOO repeats (8, 14, 15). Unlike normal families in which the repeat is stably transmitted, in fragile X families the COO repeat is unstable and tends to increase in size in 4 35 subsequent generations. Patients with fragile X syndrome have full mutation alleles of >23 repeats (sometimes> 1) which show abnormal methylation (8, 16). Nonpenetrant carriers have unstable premutation alleles of -6-2 repeats which show no aberrant methylation and are not associated with any clear phenotype (16). The risk of expansion from a premutation to full mutation size allele is dependent upon the length of the CGG repeat and has only been observed on maternal transmission (1,2,8). Repeat expansion and methylation results not only in transcriptional silencing of FMR, but also leads to delayed replication of the entire region (17) and the formation of a cytogenetic fragile site at Xq27.3 referred to as FRAXA (18). Other fragile sites have been cloned and also result from the expansion and methylation of CGG repeat tracts (19). The mechanism by which CGG repeat expansion and the subsequent methylation results in the transcriptional 7-O- 775/96/11-435$9.51 CJ 1996 Plenum Publishing Corporation.

2 436 Eberhart and Warren silencing of FMR and the fragile site formation seen in fragile X syndrome is unclear. t has been reported that CGG repeats can form unusual DNA structures in vitro (2, 21), but expanded premutation and full mutation repeats have proven refractory to cloning, thus limiting the in vitro analysis of these structures and necessitating the development of an in vivo approach. A common approach for analyzing chromatin conformation has been utilization of isolated nuclei to assess regions of nuclease sensitivity at a particular locus. ndeed, Lou et al (22) has shown that isolated nuclei from one patient with fragile X syndrome exhibits nuclease resistance at FMR. Here we extend this observation with a larger sample size and now include premutations in the study utilizing a permeabilized cell system that is less prone to loss of chromatin components that sometimes occurs during the isolation of nuclei (23). Lymphoblastoid cells from normal, premutation, or fragile X individuals were permeabilized with lysophosphatidylcholine (lysolecithin) and then incubated with a restriction endonuclease to assess chromatin accessibility. While DNA from normal and premutation cells was completely digested in vivo, DNA from full mutation fragile X males was not cleaved at the fragile locus, but was completely digested at another locus on the X chromosome. Therefore, it appears that CGG repeat expansion and methylation lead to the formation of an altered chromatin structure. MATERALS AND METHODS Cell Lines and Culture Conditions. Epstein-Barr virus-transformed human lymphoblast cells from normal males (3 CGG repeats), carrier males (84 and 14 repeats), fragile X males (from 6-1 repeats), and normal females (3 repeats) were cultured in RPM 164 media containing 1% (v/v) fetal bovine serum at 37 C in a humidified 5% CO2 incubator. Lysophosphatidylcholine Penneabilization. As outlined in Fig. 1, -17 lymphoblastoid cells were collected by centrifugation, washed with A 8 Treated Peeabi:e with Cells Lyso-PC Mspl Digestion solate DNA... Digest DNA with EcoR Southrn Analysis with probe pe5.1 Control Cells Digest DNA with Southern Analysis solate DNA..EcoR and with probe pe5.1 Mspl or Hpall Fig. 1 Schematic representation of the cell permeabilization procedure. (A) Map of the FMR region contained in clone pe5.! with key structures indicated. (8) Outline of the lysophosphatidylcholine (Lyso-PC) permeabilization protocol as described in the materials and methods. \(J

3 ty. ls!e at 'e, ld d " il T e s s h Altered Chromatin in Fragile X Syndrome phosphate buffered saline (ph 7.4), and resuspended in 1 ml of permeabilization buffer A (15 mm sucrose, 8 mm KCl, 35 rnm HEPES [ph 7.4], 5 rnm KPO4 [ph 7.4], rnm MgC2,.5 mm CaC2) (24). Cells were permeabilized by the addition of 25 J1g1ml L-a-lysophosphatidylcholine (Avanti Polar-Lipids, nc.). Permeabilization efficiency was consistently estimated at 98-1% based on trypan blue staining of an aliquot of permeabilized cells. Following a 2 min. incubation on ice, 3 ml of buffer A was added to dilute the lysophosphatidylcholine and the permeabilized cells were pelleted by centrifugation at 2 X g. Cells were resuspended in 1 ml of X React buffer (Gibco/BRL) and 1,25, or 4 units of Mspl (Gibco/BRL) were added followed by a 1 hour incubation at 37 C. Mspl was inactivated by incubation at 65 C for 1 min. and cells were lysed by the addition of 3 ml stop/lysis buffer (2 rnm Tris-HCl [ph 8], 2 rnm NaCl, 2 rnm EDTA, 1% SDS, 6 J1g1ml proteinase K) (23). Genomic DNA was isolated and treated with proteinase K and RNase. Southern Analysis. DNA from permeabilized cells was digested with EcoR and subjected to Southern analysis as described (25) using the FMR probe pe5.1 (1) which was random-prime labeled with 32P-dCTP using the Megaprime kit (Amersham). DNA isolated from control cells was digested with EcoRi alone or EcoRi followed by Hpa or Mspl and subjected to Southern analysis with probe pe5.1 as described above. To ensure the efficiency of permeabilization and Mspl digestion in fragile X patient cells, DNA from permeabilized and Mspl digested cells and control cells was digested with Pst! only or Pst! and Mspl and then isolated and subjected to Southern analysis with the M27B probe, which was random-prime labeled with 32P-dCTP using the Megaprime kit (Amersham). M27B recognizes the DXS255 locus at Xcen-pl1.22 which contains a variable copy number tandem repeat (26). RESULTS 437 n order to investigate potential differences in chromatin accessibility at the FMR locus between normal, carrier (premutation), and full-mutation fragile X individuals we utilized an in vivo approach to assess chromatin conformation (27) which was independently developed by Ymer and lans {28). This approach, in which cells are permeabilized with lysophosphatidylcholine followed by nuclease digestion, bypasses the potential problems of reproducibility and the loss of chromatin components associated with using isolated nuclei for chromatin conformation analysis (23, 28). Lysophosphatidylcholine permeabilizes the cell membrane but does not affect nuclear integrity (28) allowing the use of nuclease sensitivity assays to assess chromatin accessibility. Lyrnphoblastoid cells from patients were permeabilized by lysophosphatidylcholine and then subjected to digestion with Msp restriction endonuclease, followed by isolation of DNA and Southern analysis with probe pe5.l, which contains a portion of the 5' end of the FMRl gene including the CpG island and CGG repeat (Fig. 1). As a control, DNA was isolated from untreated cells and digested with restriction endonucleases to allow comparison of the in vivo digestion pattern with the digestion pattern ofnakeddna(fig.lb). Digestion of control (naked) DNA with EcoR alone followed by Southern analysis with pe5.l shows a 5.2 kb band in the normal males with slightly larger bands in carrier males and substantially larger bands in fragile X males due to the increase in CGG repeat size (Fig. 2A-C). Digestion of EcoR followed by Msp or its methylation-sensitive isoschizomer, Hpa, revealed the expected methylation of fragile X male DNA at the FMRl locus, with no, methylation of normal or carrier male DNA (Fig. 2A:-C) (, 29). Msp digestion gave 1.8 and 2;2 kb bands in all samples while Hpa did not cleave the fragile X male DNA, indicating

4 438 Eberhart and Warren A 1 2' Coo --N = "... on.. Uo.WWWW W 2 LYSQ-PC B 1 2 LYSO.PC LYSO-PC C oooc -"'..- "'.. ooo oo = uo.""""uo.--"" ww kb kb kb Normal Males Carrier Males c LYSO-PC LYSO.PC LYSo-PC LYSO-PC Oc Oc Oc Oc -"' ",, -"'- ",, ",, "'- ",, -"' a:=..a.a.a.a. a:= O..a.a.a.a.O..a.a.a.a.O..a.a.a.a. = = "a."""" "a."""" "a..,.,.,.",a."""" W:: WW::W kb..". 2.2 kb..". 18 kb Fragile X Males - Fig. 2 Southern blot analysis of DNA from control or ysophosphatidylcholine (LYSO-PC) permeabilized human ymphoblastoid cells from normal (A), carrier (B), or fragile X males (C) using probe pes.. Control DNA was digested with EcoR only, EcoR + Msp, or EcoR + Hpall. LYSO-PC permeabilized cells were digested with either 1, 25, or 4 units (U) of Msp as indicated and the DNA was isolated and digested with EcoR. the presence of a 5-methylcytosine at the second position in the CCGG recognition sequence of Hpa and Msp. To assess chromatin accessibility, lysophosphatidylcholine permeabilized cells were digested with 1, 25, or 4 units of Msp. These levels of Msp, which are higher than those used for isolated nuclei, are necessary for complete digestion in this intact cell system. n the permeabilized cells, DNA from normal males was cleaved by 1 units of Msp with little additional cleavage detected at 25 or 4 units (Fig. 2A). A small amount of DNA from the lysophosphatidylcholine treated cells was not cut by Msp and is visible as a 5.2 kb band. This likely reflects DNA from the 1-2% of cells which were not efficiently permeabilized as assessed by trypan blue staining. The Msp digestion pattern in permeabilized cells does not exactly match the digestion pattern of naked DNA indicating that not all Msp sites are accessible in vivo. Like normal male DNA, carrier male DNA was cleaved at all levels of Msp indicating that an increase in COO repeat number alone does not affect chromatin accessibility (Fig. 2B). However, DNA from fragile X males showed little or no cleavage even at 4 units of Msp in permeabilized cells (Fig. 2C). Since control (naked) DNA from all cell lines was cleaved by Msp, it appears there is an inaccessible chromatin conformation at the full-mutation fragile X locus caused by the repeat expansion and subsequent methylation seen in fragile X patients.

5 ren Altered Chromatin in Fragile X Syndrome 439 To further examine the role of methylation in this altered chromatin conformation the Msp digestion pattern was examined for normal, females. The FMR locus is subject to X inactivation and is methylated on the inactive X chromosome (11, 3). As expected, only about Msp digested cells was isolated and cleaved with Pst! followed by Southern analysis with probe M27B (26). M27B is located at Xcen- (DXS255) and recognizes a region which contains a variable copy number tandemrepeat. Nonnal, carrier, and full mutation malesand nonnal females showed efficient Msp ) pll.22 Y2 of normal female naked DNA was digested by Hpa due to the methylation on the inactive X chromosome (Fig. 3). nterestingly, normal : female DNA from permeabilized cells was only partially digested with Msp (Fig. 3). t appears that approximately Y2 of the DNA is digested while the remainder shows a pattern _similar to that seen in fragile X males. Therefore, the aberrant methylation associated with the expanded CGG repeat in fragile X individuals seems to result in inaccessible chromatin DSCUSSON structure similar to that which is produced by the methylation associated with X inactivation, mediated repression..to show that the chromatin inaccessibility İ 1 2 observed in fragile X males was specific to the FMRl locus, DNA from penneabilized and LYSQ-PC LYSQ-PC digestion in lysophosphatidylcholine penneabi-lized cells and naked DNA (Fig. 4). Therefore,the lack of digestion seen in fragile X males is not due to inefficient penneabilization or Msp digestion and likely represents an altered chromatin confonnation. We utilized an in vivo perrneabilized cell system to investigate the chromatin conformation at the FMRllocus. t appears that the CGG in :?:- :?:- ::) ::).:?;o th c= Coo Coo )() Lt) Lt) -?- N a. -?- N = ca a. a. a. a. = m a. a. a. a. V u Co U) U) U) U) u a. U) U) U) U) )1 )t d e W J: W ::...c;:. 5.2 kb!\ 11 :s ). d n } y n s t Normal Females Fig. 3 Southern blot analysis of control or ysophosphatidylcholine (LYSO-PC) permeabilized human ymphoblastoid cells from normal females using probe pes.. LYSO-PC permeabilized cells were digested with either 1, 25, or 4 units (U) of Msp as indicated and the DNA was isolated and digested with EcoR. kb kb

6 44 Eberhart and Warren >- T- cn Co Carrier Male, -(/) Coo +, ā. (/) _.J -J- =>..-.. (/) 2: Fragile Male _ (.) c.. n >-..J - "in =>.?:- c.. c: + Normal Female Fig. 4 Southern blot analysis of human lymphoblastoid DNA using probe M27B. Control DNA was digested with Pst! only or Pst! + Msp. Lysophosphatidylcholine (LYSO-PC) permeabilized cells were digested with 1 units (U) of Msp and the DNA was isolated and digested with Pst!. repeat expansion and methylation which results in fragile X syndrome causes an altered chromatin conformation. The potential for this altered chromatin conformation to playa role in the transcriptional silencing of the FMR gene, delayed replication of the region, or fragile site formation is intriguing. At the level of resolution provided by this Msp digestion assay, the altered conformation observed in fragile X males appears similar to that seen on the inactive X chromosome. This in vivo analysis is consistent with previous work by Luo et al. (22) which utilized isolated chromatin to show that the FMR CpO island is resistant to Msp digestion in singl fragile X male and on the inactive X chromosome. Therefore, methylation appears to be the key step in mediating this alteration of chromatin conformation. t is not clear how repeat expansion results in the aberrant methylation seen in fragile X syndrome, but the methylation level at the FMR locus correlates with the clinical phenotype of fragile X males (, 25). t will be interesting to determine how variable levels of methylation seen in some mosaic fragile X males affect chromatin conformation and how this relates to clinical involvement. One fragile X mosaic male who showed partial methylation and no cytogenetic fragile site expression demonstrated an increased sensitivity to Msp digestion in vivo when compared to other fragile X males further syggesting that methylation may be the key determinant in the observed chromatin changes (Eberhart and Warren, unpublished observation). The mechanism by which methylation is involved in altered chromatin confinnation and transcriptional suppression of the FMR gene and other loci is unclear (31, 32). t has been suggested that methylation of CpG islands can result in altered chromatin structure which either directly inhibits transcription via structural alterations (33, 34) or indirectly inhibits transcription via proteins that bind to methylated

7 Altered Chromatin in Fragile X Syndrome 441 CpG's (35). Though repeat expansion seems to precede methylation (11), further work is necessary to characterize the changes that occur following repeat expansion and methylation and to determine how these changes lead to the transcriptional silencing, delayed timing of replication of the FMRl locus (17), of the cytogenetic fragile site expression seen in fragile X syndrome. ACKNOWLEDGMENTS We thank N.J. Fraser for providjng the M27B probe. S.T.W. is an investigator of the Howard Hughes Medical nstitute. This work was partially funded by NH grant HD LTERATURE CTED 1. Warren, S.T., and Ashley, C.T. (1995). Annu. Rev. Neurosci. 18: Eberhart, D.E., and Warren, S.T. Cold Spring Harb. Symp. Quant. Bio. 61: in press. 3. Khandjian, E. W., Corbin, F., Woerly, S., and Rousseau, F. (1996). Nature Genet. 12: Tamanini, F., Meijer, N., Verheij, C., Willems, P.J., Galjaard, H., Oostra, B.A., and Hoogeveen, A.T. (1996). Hum. Mol. Genet. 5: Eberhart, D.E., Malter, H.E., Feng, Y., and Warren, S. T. (1996). Hum. Mol. Genet. n press. 6. Ashley, C. T., Wilkinson, K.D., Reines, D., and Warren, S.T. (1993). Science 262: Siomi, H., Siomi, M.C., Nussbaum, R.L., and Dreyfuss, G. (1993). Ce/l74: Fu, Y.-H., Kuhl, D.P., Pizzuti, A., Pieretti, M., Sutcliffe, J.S., Richards, S., Verkerk, A.J.M.H., Holden, J.J.A., Fenwick, R.G., Jr., Warren, S.T., Ooslra, B.A., Nelson, D.L., and Caskey, C.T. (1991). Ce/l67: Oberle, 1., Rousseau, F., Heitz, D., Kretz, C., Devys, D., Hanauer, A., Boue, J., Bertheas, M.F., and Mandel, J.L. (1991). Science 252: Verkerk, A.J.M.H., Pieretti, M., Sutcliffe, J.S., Fu, Y.H., Kuhl, D.P.A., Pizutti, A., Reiner,., Richards, S., Victoria, M.F., Zhang, F., Eussen, B.E., van Ommen, Gl.B., Blonden, L.A.J., Riggins, G.J., Chastain, J.L., Kunst, C.B., Galjaard, H., Caskey, C.T., Nelson, D.L., Oostra, B.A., and Warren, S.T. (1991). Cel/6S: Sutcliffe, J.S., Nelson, D.L., Zhang, F., Pieretti, M., Caskey, C.T., Saxe, D., and Warren, S.T. (1992). Hum. Mol. Genet. 1: Homstra,.K., Nelson, D.L., Warren, S.T., and Yang, T.P. (1993). Hum. Mol. Genet. 2: Ashley, C.T., Sutcliffe, J.S., Kunst, C.B., Leiner, H.A., Eichler, E.E., Nelson, D.L., and Warren, S.T. (1993). Nature Genet. 4: Snow, K., Doud, L.K., Hagennan, R., Pergolizzi, R.G., Erster, S.H., and Thibodeau, S.N. (1993). Am. l. Hum. Genet. 53: Kunst, C.B., and Warren, S.T. (1994). Cell 77: Fisch, G.S., Snow, K., Thibodeau, S.N., Chalifaux, M., Holden, J.J.A., Nelson, D.L., Howard-Peebles, P.N., and Maddalena, A. (1995). Am. l. Hum. Genet. 56: Hansen, R.S., Canfield, T.K., Lamb, M.M., Gartler, S.M., and Laird, C.D. (1993). Cell 73: Sutherland, G.R. (1977). Science 197: Ashley, C.T., and Warren, S.T. (1995). Annu. Rev. Genet. 29: Gacy, A.M., Goellner, G., Juranic, N., Macura, S., and McMurray, C.T. (1995). Cell 81: Fry, M., and Loeb, L.A. (1994). Proc. Natl. Acad. Sci. U.S.A. 91: Luo, S., Robinson, J.C., Reiss, A.L., and Migeon, B.R. (1993). Somat. Cell Mol. Genet. 19: Pfeifer, G:P., and Riggs, A.D. (1991). Genes Dev. 5: Taljanidisz, J., Popowski, J., and Sarkar, N. (1989). Mol. Cell. Bioi. 9: Pieretti, M., Zhang, F., Fu, Y.H., Warren, S.T., Oostra, B.A., Caskey, C.T., and Nelson, D.L. (1991). Cell 66: Fraser, NJ., Boyd, Y., and Craig,. (1989). Genomics 5: Eberhart, D.E., and Warren, S.T. (1993). Am. l. Hum. Genet. 53(suppl.):678 (abstract). 28. Ymer, S., and Jans, D.A. (1996). Biotechniques 2: Warren, S.T., and Nelson, D.L. (1994). lama 271: Kirchgessner, C.U., Warren, S.T., and Willard, H.L. (1995). l. Med. Genet. 32: Rhodes, K., Rippe, R.A., Umezawa, A., Nehls, M., Brenner, D.A., and Breindl, M. (1994). Mol. Cell. Bioi. 14: Pieper, R.O., Patel, S., Ting, S.A., Futscher, B.W., and Costello, J.F. (1996). l. Bioi. Chem. 271: Antequera, F., Boyes, J., and Bird, A. (199). Cell 62: Keshet,., Lieman-Hurwitz, J., and Cedar, H. (1986). Cell 44: Boyes, J., and Bird, A. (1991). Cell 64: