Estrogen receptor alpha (ER) is a ligand-dependent transcription factor. Upon binding estrogen, ER recruits coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes. In addition to histones, coactivators can modify ER itself and other proteins in the transactivation complex. Here, we show that ER is directly methylated at lysine 302 (K302) by the SET7 methyltransferase. SET7-mediated methylation stabilizes ER and is necessary for the efficient recruitment of ER to its target genes and for their transactivation. The SET7-ER complex structure reveals the molecular basis for ER peptide recognition and predicts that modifications or mutations of nearby residues would affect K302 methylation. Indeed, a breast cancer-associated mutation at K303 (K303R) alters methylation at K302 in vitro and in vivo. These findings raise the possibility that generation, recognition, and removal of modifications within the ER hinge region generate "ER modification cassettes" that yield distinct patterns for signaling downstream events.

Lambda exonuclease is a highly processive 5'-->3' exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by lambda exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of lambda biology, lambda exonuclease enzymology and the availability of the X-ray crystallographic structure of lambda exonuclease make it a suitable model to dissect the mechanisms of processivity. lambda Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in lambda exonuclease involved in recognizing the 5'-phosphate at the ends of broken dsDNA. The preference of lambda exonuclease for a phosphate moiety at 5' dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5'-phosphate is due to the formation of inert enzyme-substrate complexes. By examining a lambda exonuclease mutant impaired in 5'-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of lambda exonuclease has been elucidated. We propose a model in which processivity of lambda exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.

Deletion at chromosome 16q is frequent in prostate and breast cancers, suggesting the existence of one or more tumor suppressor genes in 16q. Recently, the transcription factor ATBF1 at 16q22 was identified as a strong candidate tumor suppressor gene in prostate cancer, and loss of ATBF1 expression was associated with poorer prognosis in breast cancer. In the present study, we examined mutation, expression, and promoter methylation of ATBF1 in 32 breast cancer cell lines. Only 2 of the 32 cancer cell lines had mutations, although 18 nucleotide polymorphisms were detected. In addition, 24 of 32 (75%) cancer cell lines had reduced ATBF1 mRNA levels, yet promoter methylation was not involved in gene silencing. These findings suggest that ATBF1 plays a role in breast cancer through transcriptional downregulation rather than mutations.

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Background: BRCAm and other GRm testing using next generation sequencing (NGS) in early diagnosed and/or metastatic breast cancer (BC) helps in the identification of both unambiguously defined deleterious mutations and sequence variants of unknown clinical significance (VUS). The early detection of these mutations in the proband and the family members help in risk stratification and instituting effective monitoring, surveillance and disease management strategies. Methods: Out of total 200 patients diagnosed with BC (April 2013-15) 77 unrelated individuals were consented to be profiled by NGS on MiSeq platform using TruSight Cancer panel (consisting of 94 genes including 13 genes highly associated with risk of inherited breast and/or ovarian cancer) in an IRB-approved prospective study in a CLIA compliant laboratory. Paired end sequencing was done with an average coverage of > 450X. Data was processed using STRAND software and interpreted using "Strand Omics" platform. The paired tumor samples were analysed for pathological stage, histological type and hormonal status. Results: GRm were detected in 61 cases (79%). Among all mutations detected, BRCA1/2 were found in 51% (31% in BRCA1, 20% in BRCA2) of cases. BRCA1 was found to be co-mutated with BRCA2 in 2 cases. Out of 37 deleterious mutations in BRCA1/2 genes only 10 were reported to be pathogenic (6 in BRCA1 and 4 in BRCA2) and rest were VUS. Mutation frequencies were higher among high grade IDC with HER2-ve tumors including TNBC (53%, p<0.05) with an early onset of the disease. TNBC with BRCAm were found to have no/incomplete pCR on conventional TAC regimen , subsequently started with platinum therapy and the outcome being monitored. Interestingly, 4 BRCA1 mutations including 3 non-sense and 1 frameshift mutation were found in two unrelated individuals suggesting them to be founder mutations in Indian population. The other GRm frequency (alone/ co-mutated with BRCA) was also found to be significantly high (49%) and include BRIP1, CHEK2, ERCC2, CDH1, SDHB, APC, MSH6, TP53, PALB2 and RAD51C. Stratification based on age of diagnosis(dx) showed a detection rate significantly higher in the age group of 25-50 yrs (74%) as compared to the 50-75 yrs (26%). Also a strong association of GRm status with the family history(Hx) of BC in 1st or 2nd degree relatives was indicated. Table 1: Correlation of GRm with Dx and HxGenen%Age at dx(yrs)Family Hx (Yes=Y, No=N,Unknown=UK)BRCA1193125-50 (n=23) 50-75(n=8)Y(n=13) N(n=3) UK(n=3)BRCA2122025-50(n=21) 50-75(n=9)Y(n=8) N(n=2) UK(n=2)PALB211.7>50YCHEK258.825-50 (n=4) 50-75(n=1)YATM610.525-50 (n=4) 50-75(n=2)Y(n=5) N(n=1)RAD5111.7<50Y Conclusions: Our study in a small cohort clearly highlighted the significance of germline testing and classifying the variant in larger cohort of BC patients with a strong family Hx of cancer particularly in BRCA1/2 positive families , and in women <50yrs for early detection and risk assessment. The study also indicates BRCAm to be an important contributor to the etiology of high grade HER2-/ TNBC in Indian patients. Expanded testing of this subtype is warranted to impact management of the disease with PARP inhibitors and/or platinum therapy. Citation Format: Ghosh M, ML S, Upasana M, Chodhury S, Mannan AU, Southekal S, Manjima C, Patil S, Murugan K, Mahesh B, Nayak R, Sridhar PSS, Rao N, Krishnamoorthy N, Gupta V, Satheesh CT, Subramanian K, Ajaikumar BS. Comprehensive analysis of BRCA (BRCAm) and other germline mutations (GRm) with a clinicopathological association in breast cancer: An Indian study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-06-06.