Af9/Mllt3 Interferes with Tbr1 Expression Through Epigenetic Modiﬁcation of Histone H3K79 During Development of the Cerebral Cortex

Af9/Mllt3 interferes with Tbr1 expression through epigenetic modiﬁcation of histone H3K79 during development of the cerebral cortex

Nicole Büttnera, Steven A. Johnsenb, Sebastian Küglerc, and Tanja Vogela,1

aCentre of Anatomy, Department of Neuroanatomy, University Medical Centre Goettingen, Georg-August-University, 37075 Goettingen, Germany; bDepartment of Molecular Oncology, Goettingen Center for Molecular Biosciences, Georg-August-University, 37077 Goettingen, Germany; and cDFG Research Center Molecular Physiology of the Brain at Department of Neurology, University Medical Centre Goettingen, Georg-August-University, 37073 Goettingen, Germany

Edited* by Pasko Rakic, Yale University, New Haven, CT, and approved February 21, 2010 (received for review October 21, 2009) Mutations of leukemia-associated AF9/MLLT3 are implicated in neu- methylation might be related to aging of the brain (22), the function rodevelopmental diseases, such as epilepsy and ataxia, but little is of H3K79 methylation in the development or function of the CNS known about how AF9 influences brain development and function. has not been studied in detail. However, other positions of histone Analyses of mouse mutants revealed that during cortical develop- H3 methylation mediate gene activities associated with devel- ment, AF9 is involved in the maintenance of TBR2-positive progen- opmental and cognitive functions in the CNS (23–28). The growing itors (intermediate precursor cells, IPCs) in the subventricular zone amount of data describing the implications of epigenetic mod- and prevents premature cell cycle exit of IPCs. Furthermore, in post- ifications in neurodevelopmental disorders underlines the necessity mitotic neurons of the developing cortical plate, AF9 is implicated in to investigate and understand the role of histone modifications the formation of the six-layered cerebral cortex by suppressing a during normal brain development. We report on Af9 function in the TBR1-positive cell fate mainly in upper layer neurons. We show that developing forebrain and show that Af9 expression prevents pre- the molecular mechanism of TBR1 suppression is based on the inter- mature depletion of progenitors during corticogenesis. AF9 sup- action of AF9 with DOT1L, a protein that mediates transcriptional presses Tbr1 expression in neurons and alters H3K79 dimethylation control through methylation of histone H3 lysine 79 (H3K79). AF9 at the Tbr1 transcriptional start site concomitant with decreased associates with the transcriptional start site of Tbr1, mediates levels of RNA polymerase II (RNAPolII). This study provides H3K79 dimethylation of the Tbr1 gene, and interferes with the pres- unique evidence that regulation of H3K79 methylation during ence of RNA polymerase II at the Tbr1 transcriptional start site. AF9 cortical development and differentiation of neuronal subtypes may expression favors cytoplasmic localization of TBR1 and its associa- contribute to a specific layer identity. Furthermore, this epigenetic tion with mitochondria. Increased expression of TBR1 in Af9 modification indirectly influences the NMDA receptor layout of mutants is associated with increased levels of TBR1-regulated projection neurons. expression of NMDAR subunit Nr1. Thus, this study identified AF9 as a developmental active epigenetic modifier during the genera- Results tion of cortical projection neurons. Loss of Af9 Results in Reduced Numbers of Cortical Progenitors. Because mutations of human AF9 are associated with neurode- −/− Tbr2 | histone methylation | upper layer neurons | Dot1l | Nr1 velopmental diseases, we analyzed the Af9 mouse mutant (Af9 ) −/− with regard to developmental defects of the CNS. Because Af9 F9/MLLT3 is one of multiple fusion partners of the histone mice die shortly after birth, we concentrated on embryonic and early A Af9−/− Amethyltransferase MLL1 in acute leukemia (1, 2). However, postnatal stages. As shown in Fig. 1 , the brains of P0 mice did mutations of the AF9/MLLT3 gene alone are not associated with not show any gross abnormalities, despite an improper bundling of fi leukemia but are implicated in anterior homeotic transformations callosal bers that was accompanied by a displacement of ventricular A B during mouse development (3), and in neurodevelopmental dis- cells and slightly enlarged ventricles (Fig. S1 and ). Various eases, such as mental retardation, epilepsy, and ataxia in human mouse mutants of factors involved in cortical development also display defects in the corpus callosum. We therefore judged the patients (4, 5). Little is known about AF9 function in the CNS, Af9−/− although its expression pattern implies a role during development of bundled callosal axons as evidence that a detailed analysis of mice might also potentially reveal variations in the cortical plate. the forebrain and cerebellum (6). In the mouse forebrain, Af9 is −/− Af9 embryos were smaller than control mice (Fig. S1C). transcribed in the subventricular zone (SVZ), a neurogenic com- Because Af9 is expressed in progenitors located in the SVZ of the partment that harbors progenitors for upper layer neurons (7, 8). developing neocortex, we analyzed whether cell proliferation might Af9 is also expressed in neurons dispersed over all cortical layers and be affected within the neocortex. BrdU labeling at different stages of diverges in this respect from other SVZ markers, such as Svet1 and Cux2 development revealed fewer cells in S-phase at all main stages of (6). On the molecular level, AF9 mediates transcriptional −/− B fi neurogenesis in Af9 cortices (Fig. 1 ). This loss of progenitors is activation and was classi ed as a proto-oncogene (9). The AF9 statistically significant only during later stages of neurogenesis, protein interacts with many different factors and has been impli- B C D – namely at E15.5 (Fig. 1 ) and E17.5 (Fig. 1 and ). Analyses of cated in different cellular processes (9 11). In the extensive network cell proliferation in the thymus revealed an increased number of of interacting proteins, AF9 associates with DOT1L (12, 13), the main enzyme responsible for histone H3 methylation at lysine 79 – (14 16). Dot1 is implicated in UV damage repair (17), and affects Author contributions: T.V. designed research; N.B., S.K., and T.V. performed research; S.A.J. gene expression in yeast, flies, and mammals (18, 19), whereas his- and S.K. contributed new reagents/analytic tools; S.A.J. and T.V. analyzed data; and S.A.J. tone H3 lysine 79 (H3K79) methylation correlates with gene acti- and T.V. wrote the paper. vation (20) and suppression (21). Following this line, AF9-DOT1L The authors declare no conflict of interest. complexes mediate transcriptional activation through increased *This Direct Submission article had a prearranged editor. levels of dimethylated H3K79 (13), but are also capable of tran- 1To whom correspondence should be addressed. E-mail: [email protected]. scriptional repression through hypermethylation of H3K79 at the This article contains supporting information online at www.pnas.org/cgi/content/full/ ENaCα promoter (12). Although increased levels of H3K79 0912041107/DCSupplemental.

7042–7047 | PNAS | April 13, 2010 | vol. 107 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.0912041107 Downloaded by guest on September 27, 2021 Fig. 2. Mutation of Af9 leads to a loss of TBR2-expressing cortical progenitors at E17.5. (A) PAX6- and TBR2-positive IPCs are reduced in E17.5 − − Af9 / mice. (Scale bar, 100 μm.) (B) Quantification of PAX6-positive cells in − − WT and Af9 / animals, n = 2 for both, ±SEM. (C) Quantification of TBR2- − − − − Fig. 1. Loss of Af9 results in reduced proliferation of cortical progenitors. (A) positive cells in WT and Af9 / animals. t test, n = 10 for Af9 / , n = 8 for WT, − − Nissl-stained cerebral cortex at P0 of WT and Af9 / littermates, showing no ±SEM; *, P < 0.05; **, P < 0.001. Given are the numbers of positive cells per gross abnormalities despite a displacement of ventricular cells accompanied square millimeter in the respective bin. by improper bundling of fibers (arrowhead). (Scale bar, 200 μm.) (B) During NEUROSCIENCE main stages of neurogenesis, fewer progenitors incorporate BrdU after a − − pulse at either E11.5, E13.5, or E15.5 and immunohistochemical (IHC) analyses Because we could not detect increased apoptosis in Af9 / − − − − at P0 in Af9 / , reaching statistical significance at E15.5; t test, n = 3 for Af9 / , − − cortices, we investigated the progenitors that stayed in the cell as well as controls for E11.5, n = 2 for Af9 / and controls for E13.5 and ± < −/− cycle. We labeled cycling cells with BrdU at E16.5 for 24 h and E15.5 BrdU injections, SEM; *, P 0.05. (C) Af9 progenitors incorporate A B less BrdU at E17.5 after a 30-min pulse. For statistical analyses, data of control analyzed for BrdU and KI67-positive cells (Fig. 3 and ). Loss animals from different litters were set to 100% and Af9−/− data are expressed of Af9 resulted in a significantly smaller cycling fraction (Fig. as percentage of control levels; t test, n = 4 for control and Af9−/−, ±SEM; 3C). Thus, Af9 expression is partially necessary to maintain the **, P < 0.001. (D) BrdU immunostaining at E17.5 after a 30-min pulse of pool of progenitors during cortical development. control and mutant cortices. (Scale bar, 100 μm.) Loss of Af9 Increases TBR1-Positive Neurons. It has been shown that

−/− differentiation of cortical progenitors from radial glia occurs over cells in M-phase in Af9 compared with WTlittermates, indicating IPCs to postmitotic projection neurons. This developmental process that decreased proliferation in the cerebral cortex was not a con- D E is characterized by the expression of the transcription factors PAX6, sequence of general growth retardation (Fig. S1 and ). To TBR2, and TBR1 in a sequential series that corresponds to the analyze whether Af9 mutation affected not only cell proliferation progressing development of progenitors toward a neuronal subtype but also migration into upper positions during cortical develop- (29). Because Af9 mutation led to a loss of TBR2-positive progen- ment, we injected BrdU at different time points (E11.5, E13.5, and itors, we hypothesized that the consequence would either be a E15.5) and analyzed the distribution of BrdU-positive cells at P0. − − As shown in Fig. S2, Af9 / mice displayed fewer BrdU-positive cells than control littermates, but despite their decreased numbers, these cells were mostly found in the correct positions. Mutants labeled at E15.5 showed a substantial loss of cycling cells in apical regions, indicating that the pool of progenitors might be depleted during development. Thus, Af9 mutation seemed to interfere with the proliferation of progenitors, especially during late stages of neurogenesis, but not with the capacity to migrate into the devel- opmentally determined cortical layer position. Because Af9 mutation affected proliferating progenitor cells, we further analyzed this cell population at E17.5 with PAX6 and TBR2 antibodies. PAX6-positive radial glia seemed reduced in some individuals (Fig. 2A). However, quantification of PAX6-positive cells did not reveal significant differences (Fig. 2B). In contrast, − − Af9 / brains had significantly fewer TBR2-positive intermediate precursor cells (IPCs) than WT littermates at E17.5 (Fig. 2 A and C). This loss was most pronounced at the pallial/subpallial boundary. Fig. 3. Af9 mutation leads to a premature exit from the cell cycle. (A and B) − − − Analyses of PAX6 and TBR2 expression at E15.5 did not reveal BrdU (green) and KI67 (red) double labeling of E17.5 Af9 / and Af9+/ fi −/− A B signi cant changes between Af9 and WT (Fig. S3 and ), cerebral cortex after a 24 h BrdU pulse. (C) Quantification of BrdU and BrdU/ indicating that Af9 mostly influenced progenitors during late stages KI67-positive cells, one-tailed t test, n = 2 for −/− animals, n = 3 for controls, ± of neurogenesis. SEM; *, P < 0.05. (Scale bar, 50 μm.)

Büttner et al. PNAS | April 13, 2010 | vol. 107 | no. 15 | 7043 Downloaded by guest on September 27, 2021 reduced number of differentiated neurons (e.g., TBR1-positive PCR assays of Tbr1 expression revealed that Tbr1 was up-regulated neurons) or in case of premature exit of the cell cycle, an increase in more than 2-fold in P0-derived mutant versus control brains (Fig. a subset of cortical neurons. Analyses of TBR1-positive cells 5B). Samples generated from E16.5 embryos did not show changes revealed a significant increase in the number of TBR1-expressing in Tbr1 expression levels (Fig. S5A). Taken together, these obser- cells in the mutant neocortex (Fig. 4 A and B). These TBR1-positive vations indicated that AF9-mediated regulation of Tbr1 expression cells were not only found in the lower layers (subplate and layer VI) might mainly take place in postmitotic neurons during the differ- with strong TBR1 expression, but were increased in upper layers II/ entiation from progenitors to mature neurons. III with usually lower TBR1 expression levels (30, 31). This increased TBR1 expression after Af9 knockout was corroborated by Overexpression of AF9 Suppresses TBR1 Expression. Because AF9 is Western blot analysis of proteins from control and mutant cortices not only expressed in progenitors but also in the cortical plate, we (Fig. 4C). To investigate whether other layers of the neocortex were investigated whether the modulation of TBR1 expression would affected by the loss of Af9, we performed Western blot (Fig. 4C)and depend on the AF9 protein in postmitotic cortical neurons. We quantified immunohistochemical (IHC) analyses (Fig. S3C). Af9 used an adeno-associated virus (AAV) that overexpressed a expression overlaps with Svet1 and Cux2 in the SVZ of the neo- C-terminally AU-1 epitope-tagged AF9 under the control of the cortex, two proteins involved in the generation of upper layer neu- neuron-specific Synapsin promoter to visualize the expression of rons (7, 32). We therefore reasoned that Af9 mutation might AF9 in cortical cultures generated from E17.5 WT mice. Cells that influence upper layer neuron formation similar to Cux2,where stained positive for AU-1 showed a characteristic focal nuclear mutants display an increase in the number of upper layer neurons. localization of the protein (Fig. S6). Western blot of proteins However, we found no evidence for a general change in the quantity isolated from transduced cultures after 12 days in vitro (DIV) of upper layer neurons, as indicated by SATB2 Western blot anal- showed a specific expression of AF9 as indicated by AU-1 immu- yses of mutant and control brain extracts (Fig. 4C). AF9 regulates noreactivity. Nontransduced and GFP-transduced control cells expression of CTGF in nonneuronal cells, and CTGF is expressed in did not express the approximately 64 kDa recombinant protein layer VII of the neocortex. However CTGF expression was not (Fig. 6A). Overexpression of AF9 resulted in a loss of the TBR1 affected by the loss of AF9 in mutant brains (Fig. 4C). CTIP2 is protein in a titer-dependent manner, though TBR1 expression was expressed mainly in a subset of neurons located in layer V and unaffected when a control virus expressing GFP was applied (Fig. weakly in layer VI, but its expression seemed unaffected by Af9 6A). To further prove that AF9 suppressed TBR1 expression, we mutation (Fig. 4C). In summary, these data indicated that Af9 analyzed AF9 overexpression in vitro and in vivo. In vitro, neurons mutation affected TBR2-positive progenitors, which might sub- strongly overexpressing AF9 did not express TBR1, and vice versa: sequently differentiate into TBR1-positive neurons. Although postmitotic TBR1-positive neurons were significantly increased in − − Af9 / brains, we observed no indication that other layers were affected by the loss of Af9. We therefore used BrdU labeling at different developmental stages to follow this differentiation of progenitors into TBR1-expressing neurons in mutant and control cortices. As shown in Fig. S4 A to D, Af9 mutants displayed only a slightly higher differentiation of progenitors into TBR1-positive neurons at E11.5, E13.5, and E15.5. Furthermore, real-time RT-

Fig. 5. AF9 interferes with nuclear TBR1 function. (A) AF9 overexpression results in a significant loss of nuclear TBR1 protein compared to residual cytoplasmic TBR1 protein. Quantification of TBR1 localization in AAV-AF9 or AAV- GFP transduced primary cortical neurons. ++, strong expression; +, moderate − − Fig. 4. Loss of Af9 increases TBR1-positive neurons. (A) TBR1 IHC of Af9 / expression. Given is the percentage of the number of TBR1-positive cells with − and Af9+/ cortices at P0, showing an increase of TBR1-positive cells in mutants. nuclear or cytoplasmic localization among strong and moderate AF9 or GFP Indicated is the subdivision of 10 bins for quantification (Upper), and the expressing cells, ±SEM, n =3;**,P < 0.001; ***, P < 0.0001. (B)Quantification of − − corresponding regions in an overview (Lower). (B) Quantification of TBR1- Tbr1 and Nr1 mRNA expression levels in P0 cerebral cortices of Af9 / and WT positive cells in the corresponding bins indicated in A. Given are the numbers littermates showing a mean of 2.114- (Tbr1) and 2.167- (Nr1)foldmoretran- − − of TBR1-positive cells per square millimeter per bin from WT and Af9 / ani- scripts in −/− samplesasdeterminedbyreal-timeRT-PCR,±SEM, Af9+/+ n =3, mals. n = 6 for −/− animals, n = 3 for +/+ animals, one-tailed t test, ±SEM; *, P < Af9−/− n =3;*,P < 0.05, one-sample t test with a theoretical mean of 1 (repre- 0.05. (C) Western blot analysis of proteins with different cortical layer local- senting no change of expression between Af9−/− and Af9+/+ samples). (C) − − ization showing increased TBR1 expression in P0 Af9 / . CTGF, CTIP2, and Staining of TBR1 and mitochondrial marker Prohibitin in a healthy neuron SATB2 did not show differences compared to GAPDH or β-TUBULIN. (Upper) and neuron with fragmented nucleus (Lower). (Scale bars, 10 μm.)

7044 | www.pnas.org/cgi/doi/10.1073/pnas.0912041107 Büttner et al. Downloaded by guest on September 27, 2021 strong expression of TBR1 correlated with low or undetectable TBR1 expression. As shown in Fig. 6 B and C, strong AF9 over- AF9 expression. Expression of GFP did not correlate with strong expression in vivo also correlated with weak TBR1 expression, and or weak TBR1 expression but rather showed uniform distribution. vice versa. Quantification of these in vivo results corroborated our We quantified the TBR1 expression level against the expression in vitro findings of TBR1 suppression by AF9 (Fig. 6B), showing a fi – levels of AF9 and GFP, respectively, and showed an inverse cor- signi cant increase of TBR1-negative (Tbr1 ) cells among cells relation of AF9 and TBR1 expression (Fig. S6 B and C). On the with strong AF9 (Au-1++) expression compared with cells with other hand, TBR1 expression seemed independent of the moderate AF9 (Au-1+) expression. Furthermore, we counted significantly increased numbers of moderate TBR1- (Tbr1+) expression of GFP, indicating that suppression of TBR1 by AF9 expressing cells among moderate AF9- (Au-1+) expressing cells was not a result of nonspecific effects caused by the viral trans- compared with strong AF9- (Au-1++) expressing cells. duction procedure. We next injected AAV-AF9 in the frontal brain of newborn mice and analyzed at P23 and P34 for AF9 and AF9 Directs H3K79 Dimethylation in the Neocortex. We next investigated whether AF9 represses Tbr1 transcription by direct association with the Tbr1 promoter. Cortical neurons were transduced with AAV-AF9 and AAV-GFP and after 12 DIV subjected to ChIP with nonspecific IgG or anti-AU-1 antibodies. Untreated cells served as an additional control. Real-time PCR with primers surrounding position −310, the transcriptional start site (TSS, +1), +145, and +15,000 of the mouse Tbr1 gene revealed that the AU-1 antibody specifically immunoprecipitated the AF9 protein that associated with the Tbr1 gene with an increased enrichment near the TSS (Fig. 7A). To analyze whether overexpression of AF9 affected transcriptional initiation and RNAPolII recruitment, we performed ChIP analysis of RNAPolII on different parts of the Tbr1 gene after overexpression of AF9 or intherespectivecontrols.AF9overexpression led to a statistically significant reduced association of RNAPolII with the Tbr1 TSS NEUROSCIENCE

Fig. 6. Overexpression of AF9 suppresses TBR1 expression in vitro and in vivo. (A) Western blot analysis of transduced primary cortical neurons. Transduction of AAV-AF9 or AAV-GFP (3 × 107 or 6 × 107 transforming units per 250,000 cells) results in the overexpression of the recombinant AF9, as indicated by the AU-1 band, absent in AAV-GFP-transduced cells or untransduced controls. TBR1 expression is decreased with increased AF9, although unaffected through Fig. 7. AF9 associates with the Tbr1 transcriptional start site and reduces overexpression of GFP. (B) Quantification of TBR1 compared to AF9 expression RNAPolII binding through hypermethylation of H3K79. (A) ChIP of AAV-AF9 in vivo in adult cortices (P23 or P34) transduced with AAV-AF9 at P3. Given is and AAV-GFP transduced or untransduced primary cortical cells. Antibodies the percentage of the number of TBR1-positive cells of a certain expression used were IgG and anti-AU-1. Precipitated DNA was assessed by real-time PCR level among strong and moderate AF9-expressing cells. ++, strong expression; at the transcriptional start site (TSS), and +15.000. Given is the amount of +, moderate expression; -, weak expression; –, no expression. n = 4, student’s t precipitated DNA relative to the respective input DNA ±SEM of triplicates. test; *, P < 0.05; *1, P < 0.05 (one-tailed); **, P < 0.001, ±SEM. (C) IHC analysis of Dotted line indicates the mean amplification of IgG control samples. (B) ChIP adult cortices transduced with AAV-AF9/AAV-GFP at P3. (Upper) AU-1 and of primary cortical cells transduced with AAV-AF9 and AAV-GFP or untrans- TBR1 costaining at lower and higher magnification. (Lower) GFP and Tbr1 duced. Antibodies used were IgG and RNAPolII, real-time PCR and pre- expression. In vivo, strong AF9 expression (arrowheads) correlated with weak sentation of data as in A. t-test; *, P < 0.05; **, P < 0.001. (C) ChIP of untreated TBR1 expression, and vice versa: strong TBR1 expression (arrows) correlated cultured primary cortical neurons (12 DIV) with IgG and anti-H3K79me2. DNA with weak AF9 expression. GFP expression correlated with strong (*), mod- was amplified from the positions −310, TSS, and +145, data presentation as in erate (#), or lacking (arrowhead) TBR1 expression. TBR1 expression was also A.(D) ChIP of WT cerebral cortex tissue with IgG and anti-H3K79me2. Pre- − − observed in GFP-negative cells (arrows). (Scale bars: first row, 50 μm; second sentation of data as in C.(E) ChIP of Af9+/+ and Af9 / P0 cerebral cortices with row, 20 μm; third row, 20 μm.) anti-H3K79me2 antibody, one-tailed t test; *, P < 0.05; **, P < 0.001.

Büttner et al. PNAS | April 13, 2010 | vol. 107 | no. 15 | 7045 Downloaded by guest on September 27, 2021 (Fig. 7B). These data suggested that AF9 suppressed Tbr1 expression fewer BrdU-positive cells in upper layer positions of the cerebral − − in mature cortical neurons by interfering with transcriptional ini- cortex. However, Cux2 / brains are bigger, display more BrdU- tiation and RNAPolII recruitment. positive neurons in upper layers, and have tightly packed upper Because AF9 interacts with the methyltransferase DOT1L, which layers (8). Thus, both AF9 and CUX2 differentially influence mediates di- and trimethylation of K79 of Histone H3 (H3K79me2, IPCs, where CUX2 promotes cell cycle exit at the time of upper me3), we next investigated whether the observed decrease of Tbr1 layer formation and AF9 prevents premature exit from the cell expression through AF9 activity was connected to a change in cycle and further inhibits the acquisition of lower layer identity by H3K79me2. We performed ChIP with chromatin isolated from WT suppressing TBR1 expression at the time of upper layer formation. neocortex as well as cultured cortical neurons and examined In addition to its role during proliferation, AF9 suppressed TBR1 H3K79me2 at different regions in and flanking the Tbr1 gene. As expression in mature neurons. In vitro and in vivo overexpression shown in Fig. 7 C and D, we were able to precipitate several frag- showed that strong expression of AF9 correlated with weak or ments of the Tbr1 gene from both samples using the H3K79me2 lacking TBR1 expression, thus indicating that sustained repression antibody. To analyze for differences in the H3K79me2 pattern in of TBR1 in mature neurons might be necessary for proper devel- − − Af9 / cortices, we performed ChIP from mutant and control opment of the neocortex. This suppression of TBR1 expression in forebrains. Fig. 7E shows that Af9 mutation led to a reduction of mature neurons by AF9 might also explain why AF9 is expressed in H3K79me2 at all sites on the Tbr1 gene that we investigated. These neurons over the entire cortical plate, which differs from CUX2 (6). data showed that AF9 modulated the H3K79me2 pattern at the To further investigate how AF9 might control TBR1 expression, Tbr1 locus, probably suggesting that AF9-directed H3K79me2 near we exploited the fact that Af9 interacts with DOT1L, which is the transcriptional start site suppressed Tbr1 transcription. responsible for H3K79me2 and me3. There is a strong correlation between H3K79me2/me3 and transcriptional activity (19). Our AF9 Interferes with Nuclear TBR1 Function. Our analyses of AF9 data support such a correlation with regard to the Tbr1 promoter overexpression in cortical neurons not only revealed a reduced that is subject to H3K79me2, the pattern of which was changed expression of TBR1, but indicated that residual TBR1 protein was according to the absence of AF9. At the transcriptional start site of found in the cytoplasm of the neurons. Nuclear protein was nearly Tbr1, we observed decreased methylation in Af9 mutants. undetectable in AAV-AF9-transduced neurons, while significantly Decreased methylation through loss of Af9 corresponded with more neurons retained nuclear TBR1 in AAV-GFP-transduced increased expression of TBR1. This finding of AF9-dependent cells (Fig. 5A). Cytoplasmic TBR1 has been reported in adult rodent transcriptional control via H3K79me2 supports other studies, brain and was shown to be localized to synaptosomes (33). These reporting the AF9-dependent regulation of transcription through authors proposed that synaptic TBR1 regulates expression of H3K79 methylation (12, 13). In the latter study, presence of AF9 is NMDA receptors that is dependent on the nuclear localization of also coupled to an increase in H3K79me2 at the ENaCα promoter TBR1 (34). To further analyze AF9 function in this context we and its transcriptional repression. investigated whether loss of AF9 would not only result in increased However, our analyses of the Af9 mutation indicated a general TBR1 expression but also in increased expression of NMDAR. As increase in H3K79me2 in cortical and cerebellar protein extracts shown in Fig. 5B, consistent with known TBR1 function, loss of AF9 (Fig. S5B), and we therefore speculate that genetic loci other than resulted in a more than 2-fold increase in the expression of Tbr1 and Tbr1 will also be affected through AF9-mediated H3K79 methyl- Nr1 in real-time RT-PCR assays. To further investigate the cyto- ation, possibly associated with a depression of methylation. AF9 plasmic localization of TBR1, we performed immunocytochemical mediates H3K79me2 by recruiting the methyltransferase DOT1L, stainings against a subset of synaptic markers including Bassoon, but because AF9 has an extensive network of interacting proteins, Gephyrin, NR1, PSD95, Synapsin, and Synaptophysin, but observed AF9 might also recruitother factors that exert an inhibitory effect on no colocalization with TBR1 (Fig. S5C). In contrast, we found the DOT1L activity, at least in neurons. Polycomb repression partial colocalization of TBR1 with the mitochondrial marker complexes (PRC), which inhibit initiation of transcriptional elon- Prohibitin (Fig. 5C). Although healthy cells showed partial overlap gation, have been discussed as potential counter players to DOT1L, of TBR1 with Prohibitin (Fig. 5C, Upper), the colocalization was and PRC-repressive effects might be overcome by H3K79me2/3 more apparent in neurons with fragmented nuclei, which were (19). AF9 interacts with a member of the Polycomb group, MPC3 presumably undergoing apoptosis (Fig. 5C, Lower). (11), which is also expressed in the CNS (35) and might therefore be a candidate for repressing the DOT1L-mediated methylation in the Discussion presence of AF9 in cortical neurons. PRC itself is an epigenetic In this study we have shown that AF9 contributes to the devel- factor that regulates transcription via histone modifications, such as opment of cortical neurons and thereby to the composition of the ubiquitination of H2A and methylation of H3K27 (36, 37). The six-layered cerebral cortex. In this context, AF9 controls pro- balancing of different posttranslational histone modifications may liferation of progenitors and prevents a premature exit from the be an important feature for cell fate determination. cell cycle, as indicated by fewer BrdU-incorporating cells as well as Our data clearly show that cell identity can be altered through fewer IPCs in Af9 mutants. In differentiating IPCs and their neu- epigenetic modifications during development (e.g., from a TBR1- ronal derivatives, AF9 prevents the expression of TBR1 and negative into a TBR1-positive state). We further started to analyze therefore determines their upper layer identity, as indicated by whether these changes relate to distinct neuronal functions. TBR1 increased TBR1-positive cells in mutants and less TBR1 in AF9 itself regulates the expression of NMDAR subunits in association overexpressing neurons. with CASK (34). Accordingly, increased TBR1 expression through Af9 is expressed in cells of the SVZ and in neurons located in all AF9-directed epigenetic modification resulted in increased tran- layers of the developing cerebral cortex. Studies of other molec- scription of Nr1, which might cause changed receptor composition ular markers of the SVZ, namely Svet1 and Cux2, suggested that of glutamatergic neurons. This result might potentially lead to dif- Af9 might influence upper layer formation as postulated for Svet1 ferent functions in CNS circuitry and may therefore be of sig- and shown for Cux2. A comparison of the mutant phenotypes of nificance for proper brain function. Furthermore, young neurons Af9 and Cux2 cerebral cortices shows that both genes exert use glutamate as a chemoattractant (38, 39), which specifically − − opposing effects on the pool of progenitors. Although Af9 / influences migration into upper layer positions during cortical brains have fewer proliferating cells, as indicated by markers such development. Although our initial analyses did not indicate a − − as BrdU, pHH3, and TBR2, Cux2 mutants incorporate more BrdU migratory defect in Af9 / mice, it is tempting to speculate that Af9 and have more pHH3- as well as TBR2-positive cells. As a con- mediated down-regulation of Tbr1 and specific NMDAR gene sequence, Af9−/− brains are smaller than controls and contain expression might result in a variation of NMDAR layout. Such

7046 | www.pnas.org/cgi/doi/10.1073/pnas.0912041107 Büttner et al. Downloaded by guest on September 27, 2021 variation is observed during the switch from a migratory to a post- Materials and Methods migratory neuronal phenotype. Further studies will be necessary to Primary Cortical culture, AAV Vectors, and Viral Transduction. Primary cortical further test this hypothesis. Dysregulations of NMDAR subunits are cells were isolated and transduced as described in SI Materials and Methods. also associated with different neurological and psychiatric disorders, AF9 or EGFP expression in AAV vectors of the hybrid serotype 1/2 was driven such as epilepsy (40) and schizophrenia (41), and we hypothesize by the neuron-specific human synapsin 1 promoter. AAV infections were that this dysregulation might potentially account for some of the performed at DIV3, and cells harvested at DIV12 for protein extraction malfunctions observed in patients with AF9 mutation. and immunocytochemistry. Epigenetic modifications, such as H3K4 or H3K27 methylation, have been shown to be associated with important neuronal pro- IHC, Western Blotting, and ChIP. Antibodies used for IHC and Western blot moters, such as GABAergic interneuronal Gad1, where altered experiments are listed in SI Materials and Methods. For ChIP, 2 μgof patterns are associated with schizophrenia (26). Increased inter- H3K79me2, AU-1, RNAPolII, and IgG antibodies were used essentially as neuronal differentiation has been shown after demethylation of described in SI Materials and Methods. For ChIP of cortical tissue, hemi- H3K27 at the Dlx5 promoter (23), and in adult neural stem cells spheres were dissected in 1% formaldehyde in PBS, incubated under agi- demethylation of H3K27 through MLL1 favors neuronal instead of tation for 15 min, and quenched with 1.25 M glycine for 5 min. After washes with PBS, tissue pellets were resuspended in IP buffer, homogenized with a glial differentiation through activation of Dlx2 transcription (24). To fi Dounce homogenizer, and subsequently with a syringe. Tissue pellets were our knowledge our ndings are unique in showing an implication of washed once with IP buffer and subsequently sonicated in IP buffer. After H3K79 methylation during developmental processes in general and fi fi centrifugation the supernatant was precleared with Protein A-Sepharose speci cally during neuronal differentiation and subtype speci cation and then subjected to immunoprecipitation (see Table S1 for primers used). of layer-specific neurons in the neocortex. Thus, our data contribute to the growing number of data that report on the importance of ACKNOWLEDGMENTS. We thank S. Heidrich, M. Pieper, and S. Heinzl for epigenetic modification for proper brain development and function. technical help and K. Krieglstein and P. Gruss for supporting the work.

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