Despite significant clinical and basic science advancements, cancer remains a devastating disease that affects people of all ages, races, and backgrounds. The pathogenesis of cancer has recently been described to result from eight biological capabilities or hallmarks and two enabling characteristics. These eight hallmarks are: deregulation of cellular energetics, avoiding immune destruction, enabling replicative immortality, inducing angiogenesis, sustaining proliferative signaling, evading growth suppressors, resisting cell death, and activating invasion and metastasis. The enabling characteristics are: genome instability and mutation and tumor-promoting inflammation. Survivin, the fourth most common transcript found in cancer cells, is a protein that is thought to be involved in the enhanced proliferation, survival, and metastasis and possibly other key hallmarks of cancer cells. Understanding how this gene is turned on and off is vitally important for attempt improving cancer management and therapy. Our work has identified a novel transcriptional regulator of survivin called Yin Yang 1 (YY1), which has been observed to activate some gene promoters and repress others and is gaining increasing interest as a target of cancer therapy. Our work shows for the first time that YY1 represses survivin transcription by physically interacting with the survivin promoter. Furthermore, YY1 appears to contribute to basal survivin transcriptional activity, indicating that disruption of its binding may in part contribute to survivin overexpression after cellular stress events including chemotherapy and radiotherapy.

During limb development a cluster of mesodermal cells in the distal posterior limb bud, the zone of polarizing activity (ZPA), regulates patterning along the anterior‐posterior axis. The signaling molecule responsible for this patterning is Sonic hedgehog (Shh), which is controlled by a highly conserved limb‐specific enhancer, the ZPA regulatory sequence (ZRS).ZRS microdeletion results in limbs that fail to express Shh and do not develop posterior limb elements, whereas many ZRS single nucleotide variants (SNVs) lead to anterior ectopic Shh expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. The ZRS contains putative binding sites for Hand2, Twist1, and Hoxd13: transcription factors (TFs) known to regulate Shh. Both Hand2 and Twist1 are basic helix‐loop‐helix (bHLH) TFs and are thought to function as homo‐ or heterodimers, while Hoxd13 has been shown to interact with Hand2.The present study's objective is to determine the role of these TFs in ZRS function. We hypothesize that altering Hand2, Twist1, and Hoxd13 bindings sites, either alone or in concert, will disrupt ZRS activity. To evaluate enhancer activity, ZRS was cloned into an enhancer‐GFP reporter construct. Hand2, Twist1, and Hoxd13 binding sites were altered using site‐directed mutagenesis, and constructs were electroporated into the presumptive limb buds of chicken embryos (Hamburger‐Hamilton stage 14). GFP expression was evaluated 48 hours post‐electroporation. We determined that mutating the binding sites for all three TFs in concert resulted in loss of ZRS activity. However, constructs with either Twist1 or Hoxd13 binding sites intact remained active. These data suggest that Twist1 and Hoxd13 may have either redundant or temporally distinct roles in ZRS regulation of Shh. Future work will determine whether these TFs bind the ZRS at the specific sites we have identified.

Anterior‐posterior patterning in the developing limb is regulated by Sonic Hedgehog (Shh), which is expressed in the Zone of Polarizing Activity (ZPA). Shh is regulated by a limb‐specific enhancer known as the ZPA Regulatory Sequence (ZRS). Mutations in the highly conserved ZRS have been linked to limb malformations in humans. Single nucleotide variants (SNVs) cause digit duplications, such as preaxial polydactyly (PPD) and triphalangeal thumb‐polysyndactyly syndrome (TPTPS). The ZRS mechanism is not well understood. Conditional knockout of Hand2 results in a delta‐Shh phenotype, indicating the importance of Hand2 in Shh regulation. Hand2 and Twist1 are basic helix‐loop‐helix (bHLH) transcription factors, capable of homo‐ and hetero‐dimerization, that bind E‐boxes (CANNTG) with some promiscuity. The objective of the present study was to determine whether the three ZRS E‐boxes are necessary for activity. We hypothesize that altering E‐boxes will disrupt ZRS function. Enhancer activity was evaluated using a ZRS‐GFP reporter plasmid. We generated a construct with all three E‐boxes altered using site‐directed mutagenesis. Constructs were electroporated into presumptive forelimb buds in Hamburger‐Hamilton stage 14 chicken embryos. Enhancer activity was recorded using fluorescent microscopy. Surprisingly, modification of the Hand2 and Twist1 binding sites did not affect ZRS activity. Future work is needed to identify what transcription factors are indispensable for ZRS regulation of Shh.

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The ZPA regulatory sequence (ZRS) is a limb‐specific enhancer that localizes Sonic Hedgehog (SHH) expression to the zone of polarizing activity (ZPA), which consequently regulates anteroposterior patterning in the developing limb. ZRS deletion lacks SHH expression in the limb resulting in loss of posterior limb structures, while ZRS single nucleotide variants (SNVs) can lead to ectopic SHH expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. HAND2 and HOXD13 are key transcription factors in SHH regulation that bind to the ZRS, potentially modulating SHH transcription. The present study’s objective was to determine the role of HAND2 and HOXD13 binding sites in ZRS function. To evaluate ZRS enhancer activity, ZRS was cloned into an enhancer‐GFP reporter construct. HAND2 and HOXD13 binding sites were altered using site‐directed mutagenesis, and contructs were electroporated into presumptive limb buds in Hamburger‐Hamilton stage 14 chicken embryos. GFP expression was evaluated 48 hours post‐electroporation. ZRS with mutated HAND2 and HOXD13 binding sites retained activity in the limb bud in a pattern that overlapped ZRS activity. This is unexpected since HAND2 and HOXD13 are known to be critical for SHH expression. Future work is needed to clarify the binding site(s) critical to activity and to characterize the factors involved in ZRS function. Support or Funding Information Supported in part by a grant from the Department of Pathology, Loma Linda University.