Supplementary Material for: WNT/β-Catenin Pathway and Epigenetic Mechanisms Regulate the Pitt-Hopkins Syndrome and Schizophrenia Risk Gene TCF4

<p>Genetic variation within the transcription factor <i>TCF4</i> locus can cause the intellectual disability and developmental disorder Pitt-Hopkins syndrome (PTHS), whereas single-nucleotide polymorphisms within noncoding regions are associated with schizophrenia. These genetic findings position TCF4 as a link between transcription and cognition; however, the neurobiology of TCF4 remains poorly understood. Here, we quantitated multiple distinct <i>TCF4</i> transcript levels in human induced pluripotent stem cell-derived neural progenitors and differentiated neurons, and PTHS patient fibroblasts. We identify two classes of pharmacological treatments that regulate <i>TCF4</i> expression: WNT pathway activation and inhibition of class I histone deacetylases. In PTHS fibroblasts, both of these perturbations upregulate a subset of <i>TCF4</i> transcripts. Finally, using chromatin immunoprecipitation sequencing in conjunction with genome-wide transcriptome analysis, we identified TCF4 target genes that may mediate the effect of TCF4 loss on neuroplasticity. Our studies identify new pharmacological assays, tools, and targets for the development of therapeutics for cognitive disorders.</p

Supplementary Material for: Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars

<strong><em>Background:</em></strong> Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. <b><i>Objective:</i></b> We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. <b><i>Methods:</i></b> In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. <b><i>Results:</i></b> Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. <b><i>Conclusions:</i></b> The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt

Supplementary Material for: Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars

<strong><em>Background:</em></strong> Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. <b><i>Objective:</i></b> We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. <b><i>Methods:</i></b> In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. <b><i>Results:</i></b> Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. <b><i>Conclusions:</i></b> The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt