TRIM71 reactivation enhances the mitotic and hair cell-forming potential of cochlear supporting cells.

Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming; however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 re-expression increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71's RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation.

Reactivation of the progenitor gene Trim71 enhances the mitotic and hair cell-forming potential of cochlear supporting cells.

Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming, however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 reactivation increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71’s RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation.

Homozygote loss-of-function variants in the human COCH gene underlie hearing loss.

Since 1999, the COCH gene encoding cochlin, has been linked to the autosomal dominant non-syndromic hearing loss, DFNA9, with or without vestibular abnormalities. The hearing impairment associated with the variants affecting gene function has been attributed to a dominant-negative effect. Mutant cochlin was seen to accumulate intracellularly, with the formation of aggregates both inside and outside the cells, in contrast to the wild-type cochlin that is normally secreted. While additional recessive variants in the COCH gene (DFNB110) have recently been reported, the mechanism of the loss-of-function (LOF) effect of the COCH gene product remains unknown. In this study, we used COS7 cell lines to investigate the consequences of a novel homozygous frameshift variant on RNA transcription, and on cochlin translation. Our results indicate a LOF effect of the variant and a major decrease in cochlin translation. This data have a dramatic impact on the accuracy of genetic counseling for both heterozygote and homozygote carriers of LOF variants in COCH.

Striatin Is Required for Hearing and Affects Inner Hair Cells and Ribbon Synapses.

Striatin, a subunit of the serine/threonine phosphatase PP2A, is a core member of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complexes. The protein is expressed in the cell junctions between epithelial cells, which play a role in maintaining cell-cell adhesion. Since the cell junctions are crucial for the function of the mammalian inner ear, we examined the localization and function of striatin in the mouse cochlea. Our results show that in neonatal mice, striatin is specifically expressed in the cell-cell junctions of the inner hair cells, the receptor cells in the mammalian cochlea. Auditory brainstem response measurements of striatin-deficient mice indicated a progressive, high-frequency hearing loss, suggesting that striatin is essential for normal hearing. Moreover, scanning electron micrographs of the organ of Corti revealed a moderate degeneration of the outer hair cells in the middle and basal regions, concordant with the high-frequency hearing loss. Additionally, striatin-deficient mice show aberrant ribbon synapse maturation. Loss of the outer hair cells, combined with the aberrant ribbon synapse distribution, may lead to the observed auditory impairment. Together, these results suggest a novel function for striatin in the mammalian auditory system.

Serum starvation enhances nonsense mutation readthrough.

Of all genetic mutations causing human disease, premature termination codons (PTCs) that result from splicing defaults, insertions, deletions, and point mutations comprise around 30%. From these mutations, around 11% are a substitution of a single nucleotide that change a codon into a premature termination codon. These types of mutations affect several million patients suffering from a large variety of genetic diseases, ranging from relatively common inheritable cancer syndromes to muscular dystrophy or very rare neuro-metabolic disorders. Over the past three decades, genetic and biochemical studies have revealed that certain antibiotics and other synthetic molecules can act as nonsense mutation readthrough-inducing drugs. These compounds bind a specific site on the rRNA and, as a result, the stop codon is misread and an amino acid (that may or may not differ from the wild-type amino acid) is inserted and translation occurs through the premature termination codon. This strategy has great therapeutic potential. Unfortunately, many readthrough agents are toxic and cannot be administered over the extended period usually required for the chronic treatment of genetic diseases. Furthermore, readthrough compounds only restore protein production in very few disease models and the readthrough levels are usually low, typically achieving no more than 5% of normal protein expression. Efforts have been made over the years to overcome these obstacles so that readthrough treatment can become clinically relevant. Here, we present the creation of a stable cell line system that constitutively expresses our dual-reporter vector harboring two cancer initiating nonsense mutations in the adenomatous polyposis coli (APC) gene. This system will be used as an improved screening method for isolation of new nonsense mutation readthrough inducers. Using these cell lines as well as colorectal cancer cell lines, we demonstrate that serum starvation enhances drug-induced readthrough activity, an observation which may prove beneficial in a therapeutic scenario that requires higher levels of the restored protein. KEY MESSAGES: Nonsense mutations affects millions of people worldwide. We have developed a nonsense mutation read-through screening tool. We find that serum starvation enhances antibiotic-induced nonsense mutation read-through. Our results suggest new strategies for enhancing nonsense mutation read-through that may have positive effects on a large number of patients.

Striatin is a novel modulator of cell adhesion.

The adherens junctions (AJs) and tight junctions (TJs) provide critical adhesive contacts between neighboring epithelial cells and are crucial for epithelial adhesion, integrity, and barrier functions in a wide variety of tissues and organisms. The striatin protein family, which are part of the striatin interaction phosphatases and kinases complex, are multidomain scaffolding proteins that play important biologic roles. We have previously shown that striatin colocalizes with the tumor suppressor protein adenomatous polyposis coli in the TJs of epithelial cells. Here we show that striatin affects junction integrity and cell migration, probably through a mechanism that involves the adhesion molecule E-cadherin. Cells engaged in cell-cell adhesion expressed a high MW-modified form of striatin that forms stable associations with detergent-insoluble, membrane-bound cellular fractions. In addition, striatin has recently been suggested to be a target of the poly (ADP-ribose) polymerases Tankyrase 1, and we have found that striatin interacts with Tankyrase 1 and is subsequently poly-ADP-ribosylated. Taken together, our results suggest that striatin is a novel cell-cell junctional protein that functions to maintain correct cell adhesion and may have a role in establishing the relationship between AJs and TJs that is fundamental for epithelial cell-cell adhesion.-Lahav-Ariel, L., Caspi, M., Nadar-Ponniah, P. T., Zelikson, N., Hofmann, I., Hanson, K. K., Franke, W. W., Sklan, E. H., Avraham, K. B., Rosin-Arbesfeld, R. Striatin is a novel modulator of cell adhesion.

The emerging role of lysine demethylases in DNA damage response: dissecting the recruitment mode of KDM4D/JMJD2D to DNA damage sites.

KDM4D is a lysine demethylase that removes tri- and di- methylated residues from H3K9 and is involved in transcriptional regulation and carcinogenesis. We recently showed that KDM4D is recruited to DNA damage sites in a PARP1-dependent manner and facilitates double-strand break repair in human cells. Moreover, we demonstrated that KDM4D is an RNA binding protein and mapped its RNA-binding motifs. Interestingly, KDM4D-RNA interaction is essential for its localization on chromatin and subsequently for efficient demethylation of its histone substrate H3K9me3. Here, we provide new data that shed mechanistic insights into KDM4D accumulation at DNA damage sites. We show for the first time that KDM4D binds poly(ADP-ribose) (PAR) in vitro via its C-terminal region. In addition, we demonstrate that KDM4D-RNA interaction is required for KDM4D accumulation at DNA breakage sites. Finally, we discuss the recruitment mode and the biological functions of additional lysine demethylases including KDM4B, KDM5B, JMJD1C, and LSD1 in DNA damage response.

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation.

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.