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Role of UBR7 in Cellular Differentiation

Uncovering a novel chromatin regulation pathway.

A major chromatin associated protein, which is highly conserved among eukaryotes and yet we know almost nothing about its role.

Chromatin modifiers and Autism

We conducted GO-term analysis of the genes associated autism based on genes listed in the SAFRI database. As expected the genes mutated in ASD are enriched for those with known functions related to learning and cognition (Figure X). However, genes associates with transcription and gene regulation are also highly enriched in ASD associated gene.  In fact, more than 60 chromatin-associated proteins have been implicated in the development of autism spectrum disorder (Figure X). In some cases, these genes can be readily assigned to functional groups. The telomere proteins PINX1, Tert and Terf2 have each been individually implicated in ASD, suggesting that an aspect of telomere dysfunction accounts for some aspect of ASD. Another example is the CHD8, which is mutated in several individuals with ASD (REF). Mutations in CHD8 leads to the dysregulation of genes, several of which are mutated in ASD. How the array of other chromatin-associated proteins identified may function together to produce common outcomes associated with the pathogenesis of Autism is not well understood.

Weproposethattherearefurtherfunctionalunitstobeidentifiedwithinthechromatinassociatedproteinsthathavebeenimplicated.  One of these is UBR7. Prior synthetic screens in S.pombe have identified a functional unit that also includes PHRF1, which is also mutated in autism (REF).  Our initial transcriptional analysis of UBR7 regulated genes shows that UBR7 regulates a distinct set of gene form CHD8 and therefore these pathways are distinct. In addition, of the gene mis-regulated when UBR7 function is eliminated is X and X, de novo mutations of which have been associated with ASD. ThereforewehaveidentifiedanewfunctionalunitwhenalteredcanleadtoASD.

Therapeuticimplications

Therapeutic approaches targeting epigenetic modifiers are presently under intense study for treatment of a variety of cancers and mental illness alike (REFS). Discoveries made as a result of the successful completion of this proposal will potentially translate into the rapid development of therapies for autism.

Aim1 and 2 wil determine the basic or commons aspects of UBR7 function. Since it is well conservd acrosee several single celled orgnaizems, the function of UBR7 may be

Aim3 will be the first description of the developmenta and physiological role of the UBR7 protein.

(B) INNOVATION:

The innovation associated with this application is primarily conceptual.

Identify novel pathways related to autism

Conduct synthetic lethal screens for novel interactions

(C) APPROACH:

General BACKGROUND and PRELIMINARY STUDIES

We used an unbiased BioID approach to identified novel histone H3 associated proteins. Cell lines expressed histone H3.1 that was tagged with the BirA* promiscuous biotin ligase and associated proteins that were biotinylated by H3.1-BirA* were purified on streptavidin beads and identified by mass spectrometry. Proteins biotinylated in H3.1-BirA* expressing cells were filtered against the protein biotinylated in the parental cell line and against an online list of non-specific interactors (Crapome.org). Using this approach, we enriched for known proteins associated with nucleosome and chromatin assembly function (Figure XA). One of the most prominent proteins was UBR7 (Figure X), which was not characterized previously for its association with chromatin.

UBR7 the most divergent member of a family of proteins containing a characteristic UBR-box. This domain was shown previously to acetylated N-degrons and mediate the degradation of these proteins [1]. However, UBR7 is different from other UBR family members in at least three respects; 1) the UBR-box of UBR7 does not recognize acetylated N-termini, 2) UBR7 lacks an E3-ligase domain found in other family members. Whether it facilities E3-ligase activity through the interaction with associated proteins in not known. 3) UBR7 contains a PHD domain, which is a known recognition motif for modified histones, and is not found in other UBR proteins. UBR7 is very well across conserved across metazoans, and is found in several species of fungi. In all cases the PHD and UBR-boxes show a high degree of amino acid identity.

Divergence in the UBRUBR-box and PHDdomains.

  • Crystal structures are available for the UBR-box from UBR1 and UBR2

Aim 1: IdentifythemechanismbywhichUBRaffectshistonePTMs

RATIONALE: Our preliminary data are the first to identify UBR7 as a previously undescribed chromatin associated protein that specifically recognizes H3K9 methylated histones. We show that UBR7 is associated selectively with nucleosomes containing the replication-coupled histone variant H3.1. The function of UBR7 is completely unknown. Cells lacking UBR7 are viable, but show increased levels of histone H3K79 methylation, a posttranslational modification that is associated with active chromatin. Based on the observation that UBR7 binds H3K9 methylated histones and contributes to reduced H3K79 methylation we propose that the function of UBR7 is to inhibit transcription. This aim will determine the selective recruitment of UBR7, determine what loci are specifically depend on UBR7 for H3K79 repression and determine if UBR7 is sufficient to lead to transcriptional repression.

BACKGROUND and PRELIMINARY STUDIES

UBRisa novel methylhistonebinderPHD domains are known to mediate the recognition of histone H3 amino terminal tails; but preferences for distinct modifications within H3 amino terminal tail varies between PHD domains of different (REF). Therefore, a peptide array of modified histone tail sequences was probed with recombinant full-length UBR7 (Figure X). We saw that that UBR7 consistently bound mono and di-methylated H3K9 peptides (Figure X). Phosphorylation of the adjacent Ser10 residue leads to loss of UBR7 binding. This is consistent with the pattern of other K9 methyl binding proteins such as HP1 [2, 3]. This suggests that UBR7 is removed from chromatin during S-phase when Ser10 phosphorylation is high, and binding is re-established in early G1. ITC experiments demonstrate that the UBR7 full-length protein binds the K9me3 methylated histone H3 tail with a KD of 1.03 M and does not recognize the unmodified H3.

The specificity of UBR7 for H3K9 modified histones can be seen using peptide pull-downs of endogenous UBR7 (eUBR7) from cell lysates. Biotinylated H3K9me3 modified peptides bound to streptavidin beads were able to bind endogenous UBR7 (Figure X). Histone H3K4 methylated or unmethylated peptides failed to purify eUBR7. PHD domains utilize an aromatic cage to recognize the modified H3 amino terminus (REF). Consistent with the PHD domain as the domain of UBR7 responsible for binding the histone tail, mutations of the aromatic cage eliminated the ability of UBR7 to bind histone H3 modified peptides (Figure X).  Mlo2 is the S.pombe homolog of UBR7 and contains the UBR-box and PHD domains, as well as significant homology in the uncharacterized c-terminus of the protein. Interestingly, the UBR7 protein is not found in S.Cerevisiae consistent with the lack of H3K9 methylation in budding yeast.

UBRrecognizesH3.1 andnotH3.3. The genome encodes several histone H3 variants [4]. Histone H3.1 and H3.2 differ by a single amino acid and are deposited into chromatin coincident with DNA replication. The replacement histones H3.3 and CENP-A are deposited independent of DNA replication. Histone H3.1 and H3.3 differ by only x amino acids. Standard affinity purification approaches using stable cells lines expressing either FLAG-tagged Histone H3.1 or H3.3 identify UBR7 bound to histone H3.1 (Figure x). Previously, we identified UBR7 associated with histone H3.1 using a similar TAP tag approach [5]. Surprisingly, UBR7 was absent from affinity purifications of the H3.3 histone variant (Figure x). CENP-A is much more divergent from H3.1, was also not able to associate with UBR7 (Figure X). The selective recognition H3.1 over the closely related H3.3 may be due to the ability of UBR7 to distinguish amino acids with H3.1 or it may be a result of the relative location of these two variant and their predominant posttranslational modifications. Aim X will explore the basis for the selective recognition of histone H3.1 by UBR7.

UBRlossleadstochangesinglobalhistonemethylation. In order to assess the function of UBR7 we created a UBR7 knockout HEK293 cell line by mutating the UBR7 start codon using Crispr-Cas9. Western blots show that the UBR7 protein is absent in these cells (Figure x). The UBR7 knockout cells are viable and divide with similar kinetics to the parental HEK293 cell lines. This is analogous to the phenotype of Mlo2 mutant in S.pombe, which is also viable. Protein containing histone modification-specific interaction domains are often themselves histone modifiers or bring enzymatic activity through associated protein complexes (REF). In order to determine if UBR7 affects the posttranslational modification of histones in the cell we simultaneously compared the state of 48 histone modifications in parental versus UBR7 knockout cells using a quantitative mass spec approach [6, 7]. While most histone modification states were unchanged, including H3K9 methylation, the major change we observe was in the modification status of histone H3 at lysine 79. Mono-methylation of H3K79 was unchanged; however, H3K79 di- and tri-methylation was increased 2- and 5-fold, respectively (Figure X). The majority of the increase was due to a shift from unmodified K79, to the di- and tri-methyl states. This suggest that UBR7 may link the methylation status of H3K9 to regulation of H3K79 methylation. Aim 2 will determine at what genomic loci these changes in H3K79 occur.

In yeast, the mono-ubiquitination of Lys 123 on histone H2B within the nucleosome simulates the catalysis of di- and tri-methylation by the enzyme DOT1 (REF). The E2 ligase Rad6 and E3 ligase Bre1 catalyze the ubiquitination of yH2B at this site[8, 9]. Likewise, human H2B is monoubiquitinated on Lys120 by the RNF20/40 complex, and this modification stimulates the H3K79 methylation by Dot1L1 [10], The that fact the we see a specific increase in di- and tri- methylation is intriguing, and the relationship between H2B ubiquitination and UBR7 will be determined in Aim 2.x

Background lacks a good description of where we expect to find K79me and H2Bub.  These are found at active genes…where in the gene? Check review.

K79 may be unqiue, in that there is not a demthylase ideintifed…so maybe controlling it is very improatn and only lost during replication or histone removal  so UBR7 may be preventing marks that are highly fixed!

Experimental approach

Aim 1.1 DeterminethebasisforselectiverecognitionofhistoneH3.1 versusH3.3

We will use both in vitro and in vivo approaches in order to examine the selective association of UBR7 with histone H3.1 versus H3.3. H3.1 differs from histone H3.3 by 5 amino acids (Figure X). One of these amino acids differences occurs in the unstructured tail (a.a. 31), and 4 are within the histone fold domain (87, 89,90, and 96). The amino acid differences in the histone fold determine the chaperone with which the histone variant interacts (REF).

To test whether UBR7 directly distinguishes H3.1 from H3.3 we will test the interaction of recombinant UBR7 with in vitro reconstituted H3.1 and H3.3 containing nucleosomes. We have experience in producing recombinant histones and assembling nucleosomes in vitro using salt dialysis and chaperone based assembly methods (REFS). Interactions with assembled nucleosomes and sub-nucleosome H3/H4 tetramers will be assessed using pulldown and size-exclusion chromatography. Recombinant Suv39H1 will be used to determine the effect of H3K9 methylation on binding of UBR7 to the intact nucleosome.

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Chromatin context may be the primary factor determining why H3.1, and not H3.3, selectively associates with UBR7. Histone H3.1 nucleosomes contain higher levels of H3K9 methylation relative to H3.3 nucleosomes [11]. H3.3 is found in active regions which contains H3K4methylation and is generally devoid of K9 methylation. Although H3.3 is found in telomeres and constitutive heterochromatin, which enriched in K9 methylation, and contributes to the accumulation of the mark at telomeres [12-17]. We identified the amino terminus of H3.1 as being required for binding of UBR7 to the nucleosome (Figure X). We will determine if the difference at amino acid 31 influences UBR7. We will compare UBR7 between H3.1A31S and H3.3S31A to determine if Ser 31 is necessary or sufficient to determine the H3.1 versus H3.3 binding.

Aim 1.2 Determinethemethyltransferase responsible forinrecruitmentofUBRtochromatin.

Cell type??????HEK and RPE or SHSY5Y?

There are several documented methyltransferase enzymes encoded in the human genome that are known to methylation Lys9 of histone H3. Suv39H1 and Suv39H2 are responsible for the histone H3K9 di- and tri-methylation in the pericentric and subtelomeric heterochromatin (REF). SetDB1 (a.k.a ESET) contributes to H3K9 methylation in both heterochromatin region and at repressed genes [18, 19]  G9a and GLP are involved in repression of genes within euchromatin and are known to dimerize (REF). We will test each of these histone methyltransferases individually for their importance in UBR7 recruitment using nuclear fractionation and histone H3 pulldown (Figure X) assays to assess global changes in chromatin binding of UBR7 and by ChIP-Seq for UBR7 to identify potential differential recruitment of UBR7 to different loci depending the specific methyltransferase. We will target the HMTs via shRNA or a CRIPSR approaches. Both of these approaches are standard procedures in the lab. In each case, we will validate knock-down or knockout by immunoblot. For ChIP Seq experiments we will compare UBR7 and H3K9me2 and H3K9Me3 ChIP in each knockdown/KO for to identify loci where the loss of H3K9 methylation like leads to direct loss of UBR7. ChiP-seq experiments will be conducted in collaboration with Ali Shilatifard’s lab (see letter of collaboration) with analysis assistance from Elizabeth Bartom. Quality controlled and trimmed ChIP-seq reads will be aligned to the human genome, and regions with peaks of activity will be called for each sample. Our current analysis pipeline for histone modification ChIP-seq uses the BWA aligner and the SICER peak caller [20, 21]. We will use the R package edgeR to identify differentially bound peaks between parental and UBR7 KO cells [22]. Peaks will also be annotated with information on their genomic neighborhood, and we will examine whether peaks are found preferentially in any particular genomic context using Homer [23].

Aim 1.4  DeterminetheeffectofH3K79 distributionsbyChIPseqinUBRknockoutandWTcells

Our preliminary data show that global levels of H3K79 di- and tri-methylation increase when UBR7 is lost. H3K9 mono-methylation levels remained almost unchanged, while tri-methylated H3K79 increased from 3% of the total histone H3 in the cell to 15%. Likewise, di-methylation of K79 changes from 2% to 6% when UBR7 is absent. In order to determine whether these changes occur uniformly across the genome, or whether specific loci are affected by UBR7 loss we will compare the pattern of di- and tri-methylated H3K79 in parental and UBR7 KO cells by ChIP-seq [Talk to Elizabeth about verbiage]. Since UBR7 is recruited by H3K9 methylation, we propose that UBR7 serves to suppress the formation of the H3K79 activating marks at these sites. Therefore, we expect to observe increased coincidence of H3K9me2/3 and H3K79me2/3 at sites when UBR7 is eliminated.

In order to determine if UBR7 recruitment is sufficient to suppress H3K79 methylation at a locus we will use an artificial promoter system in which UBR7 can be recruited to the locus and the resulting changes in transcriptional activity and histone PTMs can be determined. The system developed in the Reinberg lab utilizes HEK cells which contains a 5xUAS GAL4 binding site upstream of a TK promoter that drives expression of a luciferase reporter gene (Figure X)[24]. The UAS will bind the GAL4 DNA binding domain (GAL4DBD ). We will tether UBR7 to the locus by expressing a GAL4DBD-UBR7 fusion. Stable cell lines will be generated in which the GAL4-UBR7 construct under the control of a dox inducible promoter. We expect that luciferase activity will be suppressed by dox induction of the GAL4-UBR7. We will determine whether K79 methylation status changes within the promotor by ChIP using primers against the promotor region of the AP. This system will also be used to test the functional importance of the UBR-box, PHD and conserved C-terminal sequences of UBR7 in regulating transcription and H3K79 methylation.

  • Must correlate the location and H3K79 changes with UBR7 location and RNA changes!

Determine the effects of UBR7 loss on H2B ubiquitination

A potential mechanism by which UBR7 controls H3K79 methylation is by regulating the ubiquitination of histone H2B on Lys 120. The crosstalk between H2BK120Ub is a well-established mechanism by which K79 methylation is limited (REF). In order to determine whether H2B ubiquitination is affected by UBR7 we will compare the levels of we H2BK120Ub levels in extracts from UBR7 KO and parental cells by immunoblotted using antibodies specific for H2BK120Ub. We expect that if UBR7 is suppressing K79 methylation indirectly by regulating H2B ubiquitination we will observe an increase in H2BK120Ub signal in the UBR7 KO cells relative to parental cells. If we observe the expected increase in H2BK120Ub we will conduct ChIP-seq to compare H2BK120Ub occupied loci with sites where K79 di- and tri-methylation is increased. We expect that if H3K79 increase are due to H2B increased H2BK120Ub that we will observe a high concordance between loci with both H2BK120UB and H3K79me. To determine whether the changes in K79 and H2B occur within the same nucleosome we will conduct IP using anti-H3K79 antibodies from MNase treated chromatin and blot for the level of H2BK120Ub. We expect that increased K79 will results in increased levels of H2BK120Ub within the same nucleosome.

 

UBR7 may influence H3K79 methylation through the recruitment of demethylase activity or deubiquitinase (DUB) activity. In order to assess this possibility, we will purify FLAG-tagged UBR7 from stable expressing HEK293 cells (Figure X). ELISA assays will be conducted using H3K79me2/3 and H2BK120Ub containing peptides. Loss of the PTM will be assessed using modification specific antibodies. UBR7 will be purified by anti-FLAG IP and eluted with FLAG peptide. We will compare FLAG-purified UBR7 with recombinant full-length UBR7 to determine if UBR7 has any intrinsic activity. Several DUB enzymes have been identified that remove mono-ubiquitin from H2BK120, including, USP3, USP7, USP15, USP22, USP44 and USP49. [25-29]. If FLAG-purified UBR7 possess deubiquitinase activity we will test the IPs for the presences of these DUBS. Alternatively, we will identify demethylases or DUBs associated with FLAG-UBR7 by mass spectrometry of the FLAG-purified UBR7 associated complexes. We have extensive experience with affinity purification and established collaboration for this to be a successful approach.

Alternative: UBR7 Inhibits Dot1L or RNF20/40 activity?

 

Aim 2.2 DeterminetherelationshipofUBRand the PHRF1 methyltransferase inhumancells (Mlomodule)

Aim 2.3 IdentifynovelUBRinteractingpathways

In addition to the candidate approach described above, we will take an unbiased approach to identifying genes and pathways that are related to UBR7 function. We will take advantage of the fact that UBR7 knockout HEK293 cells are viable to conduct a genome-wide CRISPR-Cas9 based synthetic interaction screen. This approach will allow us to discover novel pathways involved in UBR7 function that may not be directly predicted based on domain structure of UBR7, or on preconceived ideas of the roles of H3K9 methylation. [And while we have observed synthetic interactions between UBR7 and genes identified as genetic interactors based on the MLo2 synthetic screen in S.pombe, we expect that UBR7 may take on additional roles in higher eukaryotes and therefore the range of interactions with UBR7 will be more complex.] Given how little we currently understand regarding UBR7, this approach will provide essential information about its function.

Synthetic interactions will be identified in UBR7KO HEK293 cell lines that are already established. HEK293 parental and UBR7KO cells were stably transduced with lentivirus expressing the CAS9 endonuclease and show similar rates of CAS9 activity based on cell viability using a gRNA against PSMD1, a known essential gene.  We will perform the screen as described by Doench et al [30] using the Burnello gRNA library, which targets 19,114 using 4 gRNAs per gene. We will isolate genomic DNA at day 1,7 and 14, amplify and sequence in collaboration with Ali Shilatifard’s lab (see letter of support). Analysis of the screen will be done in collaboration with Elizabeth Bartom, who will provide bioinformatics support and is a Co-investigator on this application. Reads will be mapped to a reference file of all gRNAs in the Brunello library, normalized to reads per million, and then we will determine the log2-fold change in T7 and T14 samples relative to T0 sample for each replicate. I will calculate a percent-rank for each gRNA at T7 and T14 by dividing the rank of the log2-fold change by the total number of gRNAs assigned to each replicate, then will average percent-ranks for each gRNA across replicates for each cell type. I will use the STARS algorithm to identify genes associated with gRNAs whose log2- fold change ranks are significantly lower at T7 or T14 than at T0 in UBR7KO-Cas9 but not 293T-Cas9 cells. These represent genes that caused decreased cell fitness during the screen and ultimately reduced library representation of integrated gRNAs specifically in cells lacking UBR7 expression.

Synthetic interactors will be organized into functional units using X, and functional units will be further tested for the relationship their relationship to UBR7 function. We will also compare the hits we obtain in our screen to the S.pombe Mlo2 screens performed previously and give preference to hits identified in both species. Hits in the screen will be validated by repeat the interactions individually using distinct guide sgRNAs against the gene of interest and in an independent UBR7KO cell line.

-functional tests? And downstream analysis

Conclusions and alternative approaches:

Immunofluorescence of endogenous UBR7 or expression of FLAG-tagged UBR7 does not show a characteristic pericentric heterochromatin subcellular distribution, so we do not expect that Suv39 suppression will alter UBR7 chromatin association.

Because UBR7 is highly conserved from multicellular organisms to simple fission yeast, we surmise that UBR7 plays a basic and conserved function in the cell, and therefore HEK293 cells are a reasonable model system. However, additional information regarding the role in non-transformed (RPE-hTERT) or more specified cell types, such as neuronal cells (SHSY5Y cells) may provide additional and diverse information about UBR7 function. Therefore, we will repeat the CRISPR-Cas9 screen in these cell types and compare these synthetic interactions to those observed in HEK293 cells.

Aim2: DeterminetheroleofUBRincellulardifferentiation

RATIONALE: UBR7 is an extremely well conserved protein, but is non-essential at the cellular level. Preliminary data generated in our lab shows that loss of UBR7 in neural precursor cells results in significant changes in gene transcription.  Phenotypical, loss of UBR7 influences the differentiation dynamics of iPS cell derived neuronal precursor cells. We propose that UBR7 functions normally to represses genes in the NPCs. This aim will determine more broadly the role of UBR7 in development by examining the function of UBR7 in iPS cell differentiation to determine whether if functional generally to suppress cell fate progression and determine the changes in chromatin that are dependent on UBR7.

BACKGROUND and PRELIMINARY STUDIES

UBRaffectsneuronaldifferentiation. UBR7 is strongly (although not exclusively) expressed in neuronal tissues, during both development and adulthood (Figure x). Because of the mutation in UBR7 associated with autism, we examined the effect of UBR7 genetic knockout or shRNA-mediated suppression in neuronal precursor cells (NPCs) derived from iPS cells (REF). We have found that the N124S mutation in UBR7 associated with ASD destabilizes the protein and leads to loss-of-function; therefore, we think that UBR7 knockout and suppression are a good first approximations of the effects of UBR7 in neuronal cells. Although, the specific effects of the UBR7 N124S mutation will be considered more closely in Aim 3.

NPC cells were derived from human iPS cells, and UBR7 was knocked-out by lentiviral-based Cripsr-Cas9. Knockout clones were selected by puromycin resistance. NPC cells lacking UBR7 were viable and grew with similar kinetics to wild-type cells (Figure X). However, the kinetics of differentiation between the UBR7 knockout cells and wild-type NPCs was significantly different. When NPC cells were forced to undergo differentiation [31] the cells that lack UBR7 were much faster to elaborate processes than WT cells as observe by phase imaging or by doublecortin (DCX) staining (Figure X). This phenotype was observed in UBR7 knockout NPCs and when UBR7 was suppressed in NPC cells using lentivirus encoding shRNA against UBR7 (Figure X). This suggest that the UBR7 KO cells are poised to undergo differentiation more quickly. However, from our preliminary experiments we do know if UBR7 loss leads to changes in number of or activity of the neurons that are produced. These questions will be addressed in Aim 2.x.

We propose the UBR7 is functioning to repress genes involved in differentiation of the NPCs into neurons.  Consistent with this idea we observed that NPC cells lacking UBR7 have upregulated genes that are associated with neural differentiation Gene set enrichment analysis (GSEA) of RNA-seq experiments on UBR7 NPCs compared to wild-type NPCs that have undergone differentiation identified neuronal signatures upregulated in UBR7KO NPC cells that are usually activated following NPC differentiation (Figure X)[32]. Methylation of histone H3K27 regulates neuronal differentiation, and H3K27 methylated genes are among those that are upregulated in NPC differentiation and in UBR7KO NPCs (Figure X)[33, 34]. Among the genes that are upregulated upon UBR7 loss are Neurog2 and NeuroD1 which are known to control neuronal differentiation [35, 36]. Interestingly NeuroD1 is essential for adult neurogenesis [37, 38]. Some neuronal specific genes are also upregulated, such as…..x..x.x.x These data suggest that UBR7 participates in repressing neural specific genes.

 

Preliminary data shows that the loss of UBR7 leads to abnormal cellular differentiation in neuronal precursor cells. This aim will determine broadly the physiological role of UBR7 using human iPS cells to test the specific role of UBR7 in cellular differentiation, and Zebrafish to physiological effect of UBR7 loss of function. Using iPS cells we will determine the role of UBR7 in several different differentiation pathways. We will conduct RNA-Seq and ATAC-seq to identify the gene changes that underlie UBR7s role in differentiation. Crispr-Cas9 knockout of the UBR7 protein in Zebrafish will determine the contributions of the proten to development.

 

 

-if I mention K27 in intro…show test K27 in iPS & NPC cells.

 

Aim 2.1 DeterminethecellulardifferentiationdynamicsandpotentialofUBRknockoutiPScells

 

We will generate UBR7 knockout iPS cells in collaboration with Evangelos Kiskinis, who is a co-investigator on this proposal and director of the Northwestern Stem Cell core. Dr. Kiskinis and his lab have established gene editing protocols in iPSCs using the CRISPR/Cas9 system. In order to avoid off-target effects we will utilize a double nickase approach cleaving the DNA on either side of the disease variant. By simultaneously introducing a single-stranded oligodeoxynucleotide containing the desired edit, the genomic sequence is edited by homologous recombination. After clonal purification and expansion, we will identify knockout cell lines by next generation targeted sequencing at significant depth coverage of at least 1000x. This ensures that the clones we select are 100% edited. Given the low frequency (1-4%), we typically screen at least 100 clones per targeting event. Only clones that exhibit 100% successful editing in both alleles are selected.

This is an important control in stem cell based disease modeling experiments, as genetic and potentially epigenetic heterogeneity of iPSC lines contributes to functional variability of differentiated somatic cells, confounding evaluation of phenotyping assays[39].

Can we drill down on one or a few of the major transcriptional changes and determine if they do the same thing to the NPCS.

In order to determine how UBR7 affects the ability of cells to differentiate we will examine the differentiation of UBR7KO cells on to three separated lineages, NPC, xx and xx.

Examine rescu es using UBR mutants (UBR-box, PHD, C-term and N124S).

Aim 3.2 IdentifytheunderlyingchromatinchangesdependentonUBRduringiPSand neural precursorcells

a. ChIP seq in NPC and  IPS cells (should have plent

b. RNA seq and ATAC seq at different timepoints in development.

Compare to

 

 

Aim3DeterminetheroleofUBRinorganismaldevelopment

 

RATIONALE:

 

 

Aim 3.3 DeterminetheroleofUBRindevelopmentusingtheZebrafishmodelsystem.

Functional analysis of UBR7a vs b in heterologous system (HEK rescue)

Determine loss-of-function of UBR7

Role in early development – morpholino

Role of later in later development using Cripsr Knockout

  1. Rescue with WT and PHD, UBR-box and UBR7 N124S mutant

Conclusions and alternative approaches:

RigorandReproducibility:

  • All assays will be conducted in triplicate. Appropriate statistical tests will be used to assess whether effects are….
  • We will conduct assays in at least two different cell types to determine cell-type specificity of any given outcome, or whether the observed effects are more universal.
  • The specificity of commercial antibodies against modified histones will be verified in the lab using quantitative peptide spotting assays.
  • iPS cell experiments we will examine 2 indpendnetly derived iPS cell lines.

TIMELINE:

References:

1. Tasaki, T., et al., The substrate recognition domains of the N-end rule pathway. J Biol Chem, 2009. 284(3): p. 1884-95.

2. Fischle, W., et al., Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature, 2005. 438(7071): p. 1116-22.

3. Hirota, T., et al., Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature, 2005. 438(7071): p. 1176-80.

4. Filipescu, D., E. Szenker, and G. Almouzni, Developmental roles of histone H3 variants and their chaperones. Trends Genet, 2013. 29(11): p. 630-40.

5. Foltz, D.R., et al., The human CENP-A centromeric nucleosome-associated complex. Nat Cell Biol, 2006. 8(5): p. 458-69.

6. Zheng, Y., X. Huang, and N.L. Kelleher, Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry. Curr Opin Chem Biol, 2016. 33: p. 142-50.

7. Martinez-Garcia, E., et al., The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells. Blood, 2011. 117(1): p. 211-20.

8. Robzyk, K., J. Recht, and M.A. Osley, Rad6-dependent ubiquitination of histone H2B in yeast. Science, 2000. 287(5452): p. 501-4.

9. Wood, A., et al., Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol Cell, 2003. 11(1): p. 267-74.

10. Zhu, B., et al., Monoubiquitination of human histone H2B: the factors involved and their roles in HOX gene regulation. Mol Cell, 2005. 20(4): p. 601-11.

11. McKittrick, E., et al., Histone H3.3 is enriched in covalent modifications associated with active chromatin. Proc Natl Acad Sci U S A, 2004. 101(6): p. 1525-30.

12. Udugama, M., et al., Histone variant H3.3 provides the heterochromatic H3 lysine 9 tri-methylation mark at telomeres. Nucleic Acids Res, 2015. 43(21): p. 10227-37.

13. Hake, S.B., et al., Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes. Proc Natl Acad Sci U S A, 2005. 102(18): p. 6344-9.

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