The highly conserved DYNLL1 (LC8) protein was originally discovered as a

The highly conserved DYNLL1 (LC8) protein was originally discovered as a light chain of the dynein motor complex but is Acacetin increasingly emerging as a sequence-specific regulator of protein dimerization with hundreds of targets and wide-ranging cellular functions. in human mouse and chicken cells. ASCIZ binds directly to the promoter and regulates its activity in a Zn2+ finger-dependent manner. DYNLL1 protein in turn interacts with ten binding sites in the ASCIZ transcription activation domain and high DYNLL1 levels inhibit the transcriptional activity of ASCIZ. In addition Acacetin DYNLL1 was also required for DNA damage-induced ASCIZ focus formation. The dual ability of ASCIZ to activate gene expression and to sense free DYNLL1 protein levels enables a simple dynamic feedback loop to adjust DYNLL1 levels to cellular needs. The ASCIZ-DYNLL1 feedback loop represents Acacetin a novel mechanism for auto-regulation of gene expression where the gene product directly inhibits the transcriptional activator while bound at its own promoter. proteins and its absence leads to embryonic lethality in (10 11 the only organism so far where a loss-of-function allele has been TNFSF4 analyzed. Despite its important cellular functions it remains largely unclear how DYNLL1 itself is regulated. The ATM substrate Chk2-interacting Zn2+ finger protein (ASCIZ; also known as ATMIN and ZNF822) was recently identified as a protein with dual functions in the DNA damage response and embryonic development. ASCIZ forms DNA damage-induced nuclear foci and is required for cell survival specifically in response to lesions repaired by the base excision repair pathway Acacetin (12-15) and in this context may function as a tumor suppressor of peripheral B cell lymphomas (16 17 However complete absence of ASCIZ causes late-embryonic lethality in mice (14 15 with a range of organ development defects most notably complete absence of lungs (14) that are most likely due to Acacetin DNA damage-independent functions as a Zn2+ finger (ZnF) transcription factor (14 18 Here we have sought to better understand the transcriptional roles of ASCIZ and found that it functions as a phylogenetically conserved transcriptional activator of gene expression as well as a sensor of DYNLL1 protein levels in a novel feedback mechanism for auto-regulated gene expression. EXPERIMENTAL PROCEDURES Animals and Cells Embryonic tissues and MEFs (14) human U2OS and GFP-ASCIZ-667-overexpressing U2OS cells (12) and chicken DT40 cells (13) were prepared and cultured as described. MEFs and human cells were transfected with the indicated plasmids using FuGENE 6 (Promega). For retroviral complementation the indicated constructs were cloned into MigR1 (19) virus was produced using the Phoenix packaging cell line and virus-containing medium was used to infect early passage MEFs with centrifugation at 1100 × for 90 min at room temperature. RNA Expression Array Analysis MEFs were prepared from three E12.5 embryos per genotype incubated for 48 h in a 10-cm dish of Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum with a medium change after 24 h and then transferred to a 15-cm dish for 72 h. Total RNA was extracted using an RNeasy mini kit (Qiagen). Expression profiling using standard conditions and Illumina Acacetin Mouse WG-6 v2 arrays was performed by the Australian Genome Research Facility (Melbourne). The microarray data have been deposited in NCBI Gene Expression Omnibus (accession number “type”:”entrez-geo” attrs :”text”:”GSE30417″ term_id :”30417″ extlink :”1″GSE30417). Blots Immunoprecipitations cDNAs Probes and Antibodies Northern blots using total RNA and immunoblots of the indicated protein extracts were performed as described (14). Probes were amplified by PCR from mouse genomic DNA cloned into pGEM-T and agarose gel-purified before labeling. Northern blot signals were quantified using ImageQuant software. cDNAs were cloned in the indicated vectors mutagenized using standard procedures and validated by DNA sequence analysis before use in experiments. For immunoprecipitation of endogenous proteins confluent U2OS cells from 2 × 15 cm dishes were lysed in ~2.4 ml of IP buffer (150 mm NaCl 50 mm Tris pH 7.4 0.1% Triton X-100) soluble extracts precleared using protein A-Sepharose (GE Healthcare) and 2 μg of normal rabbit IgG (DAKO) and equal aliquots were incubated with 3 μg of anti-ASCIZ or 3.