Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

School of Education

Abstract

Mixed Lineage Leukemia (MLL) is a multidomain protein whose gene is translocated in a subset of AML leukemias. Translocation of the MLL gene is present in approximately five percent of adult acute leukemias and ten percent of pediatric leukemias (Daser, A 2004, Look, A 1997, Huret, J 2001) Patients presenting in the clinic at the time of diagnosis with an MLL fusion have been shown to respond poorly to treatment and have a worse prognosis than matched wild type MLL patients (Rubnitz, J 1994, Rubnitz, J 1999). Novel therapies therefore are needed in order to more effectively treat patients with MLL leukemias.

Cyclophilin33 (CYP33) has previously been shown to be a negative modulator of MLL gene transactivation activity (Fair, K 2001, Anderson, M 2002). It has been well established that for CYP33 to exert its repressive effects on MLL several events must occur. CYP33 binds to the 3rd PHD (PHD3) finger of MLL and increases Histone Deacacetylase1 (HDAC1) to the repression domain (RD) of MLL thereby reducing target gene expression (Xia, Z 2003, Fair, K 2001, Anderson, M 2002). It has been hypothesized that cis-trans prolyl isomerase activity is important for recruitment of HDAC1 as increased CYP33 leads to increased HDAC1 at RD domain of MLL (Fair, K 2001, Xia, Z 2003, Chen, J 2008). In cells treated with the inhibitor, Cyclosporin A

(CsA), CYP33 was not able to recruit HDAC1 to MLL (Koonce, M 2004). hPC2, BMI1 and CtBP also bind to the MLL repression domain, although the role these co-repressor proteins play in suppression of MLL target gene activation has not been elucidated (Xia, Z 2003). In this study, the co-repressor proteins (HDAC and BMI1) that bind to MLL RD were examined to determine if CYP33 leads to the recruitment of HDAC1 and BMI1 to the RD of MLL. Also CYP33 PPIase activity was examined to determine if it is essential for HDAC1 and BMI1 binding to MLL.

Once HDAC1 and BMI1 were established to bind to MLL in a PPIase dependent manner, the chromatin modifications on the H3 histone tail, which are known substrates of HDAC1, were examined to determine if the H3 Acetylation (Ac) changes in response to CYP33 levels. H3Ac (H3K9Ac, H3K14Ac) and H3K27Ac are critical marks for active gene transcription, while its counterpart H3 lysine 27 trimethylation (H3K27Me3) is a mark for gene repression. It has been proposed that one of the main roles of H3K27Ac is to block H3K27Me3 (Pasini, D 2010). This hypothesis was tested in MSA cells, a thyroid carcinoma cell line, which showed robust decrease of CYP33 protein upon knockdown by siRNA treatment and concomitant upregulation of MLL target genes. Upon modulation of CYP33 in MSA cells, it is seen that H3K27Ac inversely correlates with CYP33 expression levels. H3Ac increases upon knockdown of CYP33, but does not show a reduction upon CYP33 overexpression in MSA cell line. H3K27Me3 levels are low at MLL gene promoters in MSA cells and remain unchanged. After studying the levels of H3Ac and H3K27Ac, HDAC1 and BMI1 recruitment to the chromatin was examined to determine if HDAC1 and BMI1 recruitment to MLL target gene promoters occurs in a CYP33 dependent manner. Surprisingly no change was seen upon CYP33 overexpression. A decrease of HDAC1 was seen at some MLL target gene promoters upon knockdown of CYP33 by siRNA.

After knockdown of CYP33 in MSA cells, oscillations in MLL and MYC gene expression levels were observed. This could be due to regulation of CYP33 expression by a negative feedback loop. Many such regulatory networks exist in the cell. From exploring the CYP33 that is bound to the chromatin, it is clear that CYP33 is located not only at the promoters of MLL targets, but also at the promoter of housekeeping genes, â-ACTIN or GAPDH. Also CYP33 was found to bind to the promoters of MLL and CYP33, suggesting that the oscillations could be due to a negative feedback loop.

CYP33 plays a role in the repression of MLL target genes by regulating the level of HDAC1 and BMI1 recruitment to MLL, and subsequently the levels of H3Ac and H3K27Ac. Understanding the role CYP33 plays in gene transcription leads to a better knowledge of MLL mediated transactivation and may lead to new and novel therapies in MLL leukemia.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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