Chromatin structure in postmitotic neurons might function as

Apart from its above described role in gene repression we found that peripheral H3K27me3-rich chromatin might serve as a replication barrier that prevents neuronal cell division. We could detect S-phase events in stimulated postmitotic LUHMES cells but did not find any expression of mitosis marker such as H3S10P or Cyclin B1. This indicates a block of replication either during S-phase or in G₂-phase. In many neurodegenerative diseases cell cycle events in postmitotic neurons are among the earliest signs of illness and are discussed to be responsible for the concomitant cell death observed in those diseases (144). Recent studies also found a connection between this irregular cell cycle re-activation and alterations in Tau- and Aß-levels (154,238);

two factors associated with PD and AD. Human neurodegenerative diseases are very difficult to investigate. On the one hand in vivo data can only be obtained from post-mortem brains and on the other hand data from animal models are often not transferable to humans (222). Although mouse models of AD exist in manifold variations they share a common problem. They either show a complete lack or only a mild induction of neuronal death (144,224). An interesting new approach to study neurodegenerative diseases is the use of patient-derived induced pluripotent stem cells (223). However, their generation is relatively laborious and disease phenotypes cannot always be replicated in these cell lines (225). The LUHMES cell system used in this study represents a valuable alternative to expensive or non-human model systems. The cells are of human origin, not derived from tumor tissue and we could show that they exhibit a neurodegeneration-associated phenotype upon treatment. When we stimulated proliferation in Lu d4 cells, we observed several phenomena also described in other models of neurodegeneration (223). Those include DNA replication (247), S-phase arrest (244) and replication stress (270).

However, it is largely unknown how these phenomena contribute to neuronal cell death, the hallmark of most neurodegenerative diseases. Interestingly, it has been shown that a state of cell cycle induction does not necessarily lead to an immediate cell death (224). The affected neurons seem to replicate their genome and can survive in this aneuploidy state for a long time (224). In our cell system we also observed no increase in apoptosis upon cell cycle induction. A “second hit” like inflammation or oxidative stress might be needed for a final induction of apoptosis (46). Both are also hallmarks of neurodegenerative disease. Taken together, these observations indicate a mechanism in postmitotic neurons that keeps them from completing the cell cycle and stops replication. Such an event/process could either be the loss or inhibition of a factor that promotes cell cycle progression (271) or the inhibition of replication due to an epigenetic reorganization of the genome (270). Replication fork stalling is an important function in proliferating cells. It allows the DNA-replication apparatus to stop at natural hurdles like secondary DNA structure, DNA binding proteins or DNA strand breaks (272); and more importantly it ensures the proper resumption of replication once

the impediment is gone. However, the stalling of replication is also associated with genomic instability (273). Indeed, we can deliver evidence for such an event in our cell system. We propose a model in which the restrictive chromatin environment at the nuclear periphery of neurons represents a replication barrier that prevents cell cycle progression into M-phase. This nuclear neighborhood is characterized by an accumulation of heterochromatin-specific histone modifications like H3K27me3 and H3K9me3 that might be coupled to the nuclear envelope. Since we only observed this phenotype in postmitotic cell types this model could help to explain the absence of postmitotic cancer cells and the cell cycle-related events that can be seen in many neurodegenerative diseases.

The activation of cell cycle after differentiation of LUHMES cells could also prove to be a useful model to test other hypotheses like for example a two-hit model in which a pre-established, non-lethal phenotype (e.g. cell cycle activation) leads to apoptosis when combined with a second insult (e.g. oxidative stress)(46).

5.6 Supplementary figures

Figure S9: List of antibodies

Antigen Host Supplier & Cat. No.

H3K4me3 Rabbit Active Motive, 39915 H3K4me3 Rabbit Millipore, CS 200580 H3K9Ac Rabbit Active Motif, # 39918 H3K9Ac Rabbit Cell Signaling, # 96715 H3K9me3 Mouse Active Motif, No 39286 H3K27me3 Rabbit Millipore, 07-449 Ki67 mouse BD 5136525X CDKN1B Mouse IgG1 BD 610242

Tuj1 mouse Covance Research Products Inc; MMS 435P PCNA mouse IgG2a Millipore; MAB424

H3S10P Rabbit Cell Signaling, # 9701 CCNA1 mouse mono Santa Cruz,sc-271682, IgG2A CCNB1 Rabbit Cell Signaling, #4138 yH2Ax mouse IgG1 Millipore;05-636 GAPDH mouse invitrogen; 39-8600

Figure S10: diagram showing KNIME-based image processing and segmentation of nuclei

Figure S11: List of PCR primers

genomic DNA Primers

gene forward reverse

AADC TCAGGATTCCCAGAGGCGA GCTGAGGCACTCGGCACTGAG BDNF AGCCCAACAACTTTCCCTTT GAGAGCTCGGCTTACACAGG BMP7 GCCCCTCAGTCCCTGTATC GTGGTGAGTGGGGAGAGGT CCNA2 GTTGCCCAGCCTTTAGCTC AGAAAACGGAGAATCGGAGAT

CCNB1 GTCGCTGAGCTTCAGTTCCT CTACGGTAGCAGCAATAATATAGTTCA CCND1 TCCCTCCTAGCTGTCCTCCT CGGACTGCTTCTCTCCAAAC CCND2 TGTCTGAGGTCACCCCATC CCAAAATACGACCCTCCAAC CDNK1C CCACCCTGCCCAGTAGATG ACCCAGTACAACAGCTTCCAG DCX CAAAGACACTGGCTGTTCCCTG CTACCCAAGGTTAGCATCTCCA DRD2 CCAGGACCAGAAGTTTGTGCG AGA AGG AGG GAG AGG GGA TAG C EZH2 ATTTCTGCGGGAAGCTACAA TCTTGGCTTTAACGCATTCC FOXD3 GTAGCGAGCGCCTAGTACC CTGGGCTCAGCTCACACTC FOXG1 GGCGGTGGTTGTTTCTTTT GCTGGCAAGTCATGTAGCAA GAPDH CCCAAAGTCCTCCTGTTTCA TAGTAGCCGGGCCCTACTTT GBX2 TGCTCCTCCTTTCTTTCCTTC CTGACCCCAGGAAGAGAGTG HES5 CGCATCAACAGCAGCATCG CTGACAGCCATCTCCAGGATGT KCNQ5 TCTGTCCACTGAACTGCTGAG CCCTGCCTTCCAGGTATTATC KIF14 TTTGCCCCCAAGTTAATCAT TCTCACAAGATTTTCAGGACTGTT KLF10 GGCGAGGCATGTGAACAAA GCTCAGGAAGTAGGGGAAAGG MYOD1 CAAATCAGGGGACAGAGGAG TTCCAAACCTCTCCAACACC NGN2 GGGGCAGATCTGATTGTTTT ACTCCCAGGCACTCCAGTTA NURR1 CGTCAGGTCGGAAATATACCA CTGCCAACATGCACCTAAAG OLIG1 TTAGCGACCTGAACCTGGC TGATGGACATTTGGGGTGCT OTX2 CAGCAAATCTCCCTGAGAGCGG GAGGAAGGCGGCTAGAGTTCTAAAC RPL30 TGAAGAGCTTTGCATTGTGG CGGAGTTACAAATGGCAACC PAX3 GCTGTCGGTTCCTAGTCCAG CTGGAACATTTGCCCAGACT PITX3 AGGCTCACTCCCTCCGAGAG GCAACAGGCAGACTCCCAGTA SHH ATGCTGCTGCTGGCGAGAT CTGCTTGTAGGCTAAAGGGGTCA SIX2 AAGAGGGAGGGAGGAGGAG CACCCCCTTAAACGGTCTC SNCA GCTTGCTTCTCCATTCTGGTGTGA CCCCCTTTTCTCTCCCCCC SYN1 AGCGGAGGAGTCGTGTCGT ATGTACCCATTTGGCAGATTGGC T GGACCTTCCTTTTCCAGATGGTGA CCTCCGATGCCTCAACTCTCC TUBB3 CCCTTCCGAGCTCTGATCC CTGAGCTTTTGCCGGTTTT WNT8B TCTACCCCCACTGGAGTCAA GCGAAGACTTTTCCCAAACA cDNA Primers

gene forward reverse

AADC GAGTCACTGGTGCGGCAGGA CCGTGCGAGAACAGATGGCA BDNF GCAGGGACCAGAAGCGTGAC TGAAATAACCATAGTAAGGAAAAGGAT BMP7 AGGCCTGTAAGAAGCACGAG GGTGGCGTTCATGTAGGAGT CCNA2 AGAAGATGAAAAGCCAGTGAGTG CCAGTCCACGAGGATAGCTC

CCNB1 TGGTGCACTTTCCTCCTTCT CAGGTGCTGCATAACTGGAA CCND1 TGAGGCGGTAGTAGGACAGG GACCTTCGTTGCCCTCTGT CCND2 ATGTTCCTGGCCTCCAAAC GCCAGGTTCCACTTCAACTT CDNK1C TTCTCAGGCGCTGATCTCTT AGCTGCACTCGGGGATTT DCX GCGAAATTTTTCAGGACCAC CACAGAAGCCATCAAACTGG DRD2 ACGGCGAGCATCCTGAACTT GCCGGGTTGGCAATGATGCA FOXD3 CTCCAGTACCCGTACGCGCT ACTTGATGAGCGACGCGGTG FOXG1 AGAAGAACGGCAAGTACGAGA TGTTGAGGGACAGATTGTGGC GAPDH ATGGAGAAGGCTGGGGCTCA AGTGATGGCATGGACTGTGGTCAT GBX2 CTCGCTGCTCGCCTTCTC CGGGTCTTCCTCCTTGTGAG HES5 AGAGAAAAACCGACTGCGGAAGCC CGACGAAGGCTTTGCTGTGCTTC HPRT1 GCTATTGTAATGACCAGTCAACAGGGG GGTCCTTTTCACCAGCAAGC KCNJ6 TTCCTTCCCTCGCCATCCGT CCACTGGGCTTTCGACGTCC KCNQ5 TTCATGTTGCAAAACGGAAG GCCCTTTTCCAAGAATTTGA KIF14 TGGTTAATCGTGCTCCAGAA ACTGGCCAAGTTGCGAATA KLF10 ATCTGCTTCCGGGAACACC TTGAAAGGTGCGGCAATGTG NGN2 CAGGCCAAAGTCACAGCAAC CCGAGCAGCACTAACACGTC NURR1 TGCTGCCCTGGCTATGGTCA AATGCGCTGTAGCCCCTGTG OLIG1 GCGGTTGGTTTTCGTTTTTA CCAGTGTTTTGTCGCAGAGA OTX2 GTTCAGAGTCCTTGGTGGGT CCCTCACTCGCCACATCTAC RPL13A GGTATGCTGCCCCACAAAACC CTGTCACTGCCTGGTACTTCCA RPL30 TGAAGAGCTTTGCATTGTGG CGGAGTTACAAATGGCAACC PAX3 GACTGGCTCCATACGTCCTGGTGC CGGCTGATGGAACTCACTGACGG PITX3 CTTGCAGCCGTGCTCTG TACTGGGAGTCTGCCTGTTG SHH CTCGCTGCTGGTATGCTCG ATCGCTCGGAGTTTCTGGAGA SIX2 CAGGTCAGCAACTGGTTCAA CCGATGGAGTCTTCTCATCC SNCA CAGGGTGTGGCAGAAGCAGC TGCAATGCTCCCTGCTCCCT SYN1 TCAGACCTTCTACCCCAATCA GTCCTGGAAGTCATGCTGGT T CAA ATGCGCTCGTCCCTGGG CGAAGCCCAGACTCGCTACC TUBB3 AACTACGTGGGCGACTCGGA GTTGTTGCCGGCCCCACTCT WNT8B GGAAATTGTTCACCGACCAG CCGTCTTGCTTACCCATCTC

Figure S12: PCA analysis of Lu d0, Lu d3 and Lu d6 microarray samples

Figure S13: Western blot analysis of different histone modifications in Lu d0 and Lu d6

Figure S14: Immunofluorescence stainings cell cycle-related markers Lu d6 cells were generated from Lu d0 and both were stained with antibodies specific for the cell cycle markers PCNA (A), H3S10P (B), CCNA1 and CCNB1 (C).

Nuclei were stained with the DNA dye H-33342 (blue). Scale bars: 50 µm.

Figure S15: Quantitative analysis of PM-treatment via EdU-pulse

All cells were treated with 5µM EdU for 30 min. EdU was added either directly before cell fixation or 2 days before (“pulse”). In case of “pulsed” cells, plates were washed two times with PBS after treatment, then fresh medium was added. After fixation cells were stained with an antibody specific for cyclin A1. The overlapof positive cells for both EdU and Cyclin A1 was calculated and is displayed as percentage of all cells.