Absence of Nsd1 alters expression of known GATA1 complex forming proteins

As shown before, the lack of Nsd1 influenced regulation of known GATA1 target genes: repressed GATA1 target genes were downregulated in Vav1-iCre;Nsd1fl/fl whereas activated targets did not increase to the same extent as seen in Nsd1fl/fl control cells (Figure 34). Interestingly retroviral overexpression of Gata1 induced the expression of erythroid differentiation markers resulted in erythroid maturation. Previous studies have shown that GATA1 is part of a protein-protein interaction network controlling transcription of erythroid differentiation regulating genes. Depending on the partner, it can form repressing or activating transcription factor complexes 63,65. One of these protein interaction networks is the activating pentamer containing T-Cell Acute Lymphocytic Leukemia 1 (TAL1, also known as SCL), Transcription Factor 3 (E2A), LIM Domain Binding 1 (LDB1) and LIM Domain Only 2 (LMO2) (see Figure 36A for schematic overview) 214. The LIM (Lin11, Isl-1, Mec-3) domain is protein- protein interaction motif presenting as tandem zinc finger 215,216. Together the complex binds to erythroid- differentiation associated regulatory sequences such as the globin locus 217.

We hypothesized that the lack of Nsd1 may result in aberrant GATA1 protein containing complexes being “repaired” upon overexpression. To obtain a first insight into formation of this GATA1 complex, we used an immunoprecipitation (IP) strategy. First we tested the efficiency of different protocols for preparation of cytoplasmic and nuclear extracts using five to ten times sonication and increasing amounts of benzonase (5-10U/µl) on murine erythroleukemia cells (MEL), which we previously used as a positive control for GATA1 IF and western blots. Increased amounts of benzonase (10U/µl) and 5 sonication steps allowed us to immunoprecipitate increased amounts of chromatin bound nuclear GATA1 (data not shown). Next, I tested the optimal antibody concentration for immunoprecipitation of GATA1 in MEL cells. Using a protocol to mildly separate cytoplasmic from nuclear fraction, we could achieve enrichment of nuclear signal (Figure 36B) 218. However, standard mild nuclear lysis, including sonication and benzonase treatment, was not able to fully free GATA1 off the chromatin. We then prepared nuclear extracts from control and Vav1- iCre;Nsd1fl/fl differentiating proerythroblasts and first checked the expression of some of the best studied GATA1 complex members. As expected, GATA1 protein levels were significantly increased in Vav1-iCre;Nsd1fl/fl cells compared to Nsd1fl/fl controls.

FIGURE 36. Immunoprecipitation of GATA1.

(A) Schematic depiction of elected complex members of GATA1. GATA1 forms a pentamer with FOG1, LMO2, LDB1, SCL and E2A, generally associated with activation of transcription. Moreover it can also form repressive complex, e.g. with ETO2. (B) Immunoprecipitation of GATA1 using N6 antibody in MEL cells. GATA1 was detected in western blot with D52H6 antibody. (C) Western Blot of nuclear extractions (10ug) of Nsd1fl/fl and Vav1-iCre;Nsd1fl/fl erythroblasts for abundance of factors. (D) Immunoprecipitation of GATA1 using N6 antibody in Nsd1fl/fl differentiating erythroblasts. GATA1 was detected in western blot with D52H6 antibody. (E) Immunoprecipitation of GATA1 using N6 antibody in Vav1-iCre;Nsd1fl/fl differentiating erythroblasts. GATA1 was detected in western blot with D52H6 antibody.

Interestingly, protein levels of the activating complex such as SCL, LDB1 and E2A were decreased and repressing complex member ETO2 was increased in Vav1- iCre;Nsd1fl/fl nuclear extracts (Figure 36C). Using the optimized conditions, we aimed to precipitate GATA1 in differentiating control erythroblasts. However, we were not able to pull down enough protein to perform qualitative analysis of the complexes. Most of the protein was lost in cytoplasmic extracts or appeared to be tightly bound to chromatin (Figure 36D & data not shown). Unfortunately, control cells are not as stable in liquid culture as Vav1-iCre;Nsd1fl/fl erythroblasts therefore yielding first lower cell numbers and second smaller cells during differentiation and therefore less protein for further analysis. Next we decided to use only Vav1-iCre;Nsd1fl/fl cells, that express higher GATA1 protein levels. We could successfully pull- down GATA1 in these cells (Figure 36E). As we miss the appropriate controls for this experiment we are currently exploring MEL cells with Nsd1 knockdown to dissect the influence of the GATA1-complex (ongoing). Taken together, our observations suggest that the absence of Nsd1 results in formation of an aberrant GATA1 containing complex: establishing efficient immunoprecipitation protocols will help to dissect these complexes in MEL cells (ongoing).

FIGURE 37. Retroviral overexpression of full-length and NSD1 mutants in pro- erythroblasts of diseased Vav1-iCre;Nsd1fl/fl mice.

(A) Schematic depiction of experimental setup using lineage depleted bone marrow cells of Vav1- iCre;Nsd1fl/fl that were kept in maintenance medium for more than six days to create stable ESRE cells. Cells were transduced in maintenance medium with pMSCV-pgk-puro-IRES-GFP, pMSCV-mNsd1-pgk- puro-IRES-GFP, pMSCV-mNsd1-ΔNID-pgk-puro-IRES-GFP, pMSCV-mNsd1- ΔNID /Setmut-pgk-puro- IRES-GFP and pMSCV-mNsd1-Setmut-pgk-puro-IRES-GFP constructs, EGFP+ cells sorted and kept under puromycin selection in methylcellulose M3434. (B) Percentage of EGFP+ cell population two days after pMSCV-pgk-puro-IRES-GFP or pMSCV-mNsd1-pgk-puro-IRES-GFP transduction of Vav1- iCre;Nsd1fl/fl ESRE cells. (C) Relative number of colonies formed in methylcellulose M3434 of Vav1- iCre;Nsd1fl/fl ESRE cells transduced with mNsd1 wildtype and mutants compared to mock transfected cells. (D) Quantitative real time PCR of Nsd1 exon 5 after transduction of ESRE cells with mNsd1 wildtype and mutants. Ct values were normalized to Gapdh expression and shown as relative expression using 1/dCt method (n=2). Data presented as mean, error bars represent ±SD.

4.3.16

Attempts to rescue erythroid differentiation of

Vav1-

ICre;Nsd1

erythroblasts by overexression of Nsd1

To demonstrate that blocked erythroid differentiation in Vav1-iCre;Nsd1fl/fl cells is indeed the direct consequence of the lack of Nsd1 we tried to restore its activity by expression of wildtype or different Nsd1 mutants. We have generated retroviral constructs (pMSCV) expressing full-length Nsd1 and mutants lacking either the nuclear receptor interacting domain (NID), the SET domain or both. Proerythroblasts kept in maintenance culture for more than six days were transduced with either the empty vector (pMSCV-pgk-puro-IRES-EGFP = mock) or full-length Nsd1 or the different mutants: mNsd1-ΔNID, mNsd1-ΔNID/ΔSETmut or mNsd1-ΔSET. We selected transduced cells with puromycin selected and plated sorted EGFP+ cells into methylcellulose (Figure 37A). Although we observed a decent transduction efficiency of about 20% with to control vector, transduction with any of the Nsd1 constructs was very poor reaching less than 1% (=1500 living EGFP+ cells per 106 starting cells) (Figure 37B). The size of the Nsd1 cDNA of about 8000bp seemed to significantly impair generation of viral particles. Nevertheless plating the small number of selected cells revealed significantly reduced colony formation upon transduction of wildtype or mutant Nsd1 compared to mock transduced cells (Figure 37C). Albeit the limitation of the small number of cells, it seemed that cells formed more colonies when transduced with the mutant than the wild type Nsd1 expressing virus. Quantitative RT-PCR revealed higher Nsd1 expression in transduced cells compared to controls (Figure 37D). Although limited by poor viral gene transfer these observation indicate that overexpression of Nsd1 significantly impairs colony formation of Vav1-iCre;Nsd1fl/fl proerythroblasts. Further work by expressing smaller truncation mutants might be necessary to dissect the critical domains that are responsible for the effects.