Influence of FOX genes on aging and aging-associated diseases. Elena Tschumak

in FOXP1 and FOXP2 SUMOylation.

      Influence of FOXP2 SUMOlyatin on various diseases It is known that not only FOXP1 and FOXP4 transcription factor TBR1 and co-repressor CtBP1 interact with FOXP2. (Deriziotis et al., 2014; Li et al., 2004; Meulmeester and Melchior, 2008; Rytinki et al., 2009). So by speech and language disorders afflicted family showed a strongly reduced FOXP2 SUMOylation. SUMOylation appears to occur in thousands of nuclear proteins in all cell types at different stages of development (Meulmeester and Melchior, 2008; Gwizdek et al., 2013). According to Bacon and Rappold (2012) and Zhao et al. (2014) SUMO influences dendritic and synaptic morphogenesis by modifying neuronal proteins. Different PIASs may be involved in SUMOylation of various FOXPs and may affect the modification of neuronal circuits. In addition, they support the neuronal plasticity, but it is unclear whether a change in SUMOylation level during brain development leads to functional impairment. It has been demonstrated that PIAS1 knockout causes perinatal lethality in mice. (Loriol et al., 2012; Hasegawa et al., 2014) The study of Estruch et al. (2016) revealed that the four PIAS were continuously expressed but the PIAS1 and PIAS3 were expressed at the same extent in all tissues tested while the PIAS2 and PIAS4 were expressed primarily in testis. Studies of Turner et al. (2013), Adegbola et al. (2015), Nazaryan et al. (2014), Utine et al. (2014) and the fact that the FOXP2 plays a role in neuronal migration, neurite growth, in synaptic plasticity and in the language-related brain circuits (Garcia-Caleroet al., 2015; Tsui et al., 2013; Verne et al., 2011; Groszer et al., 2008) allowed another hypothesis about the cause of speech disorders in the KE family. All members of the by the speech disorder afflicted KE family had a heterozygous R553H mutation in the DNA-binding domain. In twenty other similar cases a missense mutation (R553H) in the FOX DNA binding domain that makes DNA binding and transcriptional repression by FOXP2 impossible was found. Nonsense and frameshift mutations, as well as chromosomal rearrangements were accompanied by similar disabilities. In contrast to the partially compensated interactions between the SUMOS and the K674R mutant the BRET assay showed an almost complete loss of the interactions between the SUMOS and R553H mutant compared to the wild type. The most important SUMOylation site stayed intact in the R553H mutants so the reduction of SUMOylation may base on reduced or absent interaction between the mutant and the PIASs or UBC9. In contrast to the K674R mutants which interact with PIAS but cannot be SUMOylated, the R553H mutants had reduced interaction with PIAS, which can be compensated by PIAS overexpression or by a fusion with UBC9. The R553H mutation also led to partial mislocalization and increased protein aggregation. The R553H mutation may lead to a conformational change that blocks the PIAS binding site. Alternatively, the loss of DNA binding capacity resulting from the R553H mutation and / or destabilization of the protein domain may trigger FOX interactions with other cellular proteins that interfere with PIAS binding. It seems to be useful to investigate the extent of the FOXP2 SUMOylation and the FOXP2-PIAS interactions in different issues and possibly further regulatory mechanisms of aging related neurodegeneration.

      FOXP2 regulates proto-oncogenes p21WAF1 / RAS 1, BCL-2, HES1 and other cancer-relevant genes. Gascoyne et al. (2015) demonstrated in „The forkhead transcription factor FOXP2 is required for regulation of p21WAF1 / CIP1 in 143B osteosarcoma cell growth arrest“ with the help of human osteosarcoma cell cultures and normal human osteoblasts that the forkhead transcription factor FOXP2 regulates proto-oncogenes p21WAF1 and CIP1. These proto-oncogenes are required for osteosarcoma cell growth arrest. lncRNA-p21 influences cell proliferation and carcinogenesis via vβ-catenin and JunB. The lncRNA HULC is highly expressed in age related colorectal carcinomas and UCA1 is highly expressed in bladder transitional cell carcinoma by modulating p27 level and can affect cellular senescence.(Parket al., 2011; Smith-Vikos and Slack, 2012) The decrease of growth factor level as well as MAPK reduction induces FOXP2 expression. In turn FOXP2 expression triggers cyclin-dependent kinase inhibitor p21WAF1 / CIP1 expression. It is known that p21 is one of the p53 target genes. (Deiry et al., 1994)19ARF/p53 and p16INK4a/Rb stop cancer cells from proliferation and play an important role in cellular senescence.

      During these experiments, the DMEM-cultured and ATCC-derived osteosarcoma cell lines were treated with the IKK pathway inhibitor VII, the MAPK pathway inhibitor, the LY-294002 PI3K pathway inhibitor, the 117082 NF-kappa B pathway inhibitor, the Notch signalling pathway inhibitor or with the DMSO vesicle. Then the alkaline phosphatase and MTS assays were done and 405nm absorbance was measured. Other methods were RNA isolation, cDNA preparation and real-time PCR, protein isolation, immunoblotting, chromatin immunoprecipitation and flow cytometric analysis of the cell cycle distribution. The authors found a link between the 7-time increased FOXP2 expression in murine bone 48 h after treatment and the decreased growth of osteosarcoma cell lines. Unlike mesenchymal tissue osteoblasts did not require RUN involvement in FOXP2 induction. (Zhao et al., 2014) In neuroblastoma cells growth arrest was not accompanied by FOXP2 induction. Cyclin-dependent kinase inhibitors such as p21WAF1, p27KIP1, CIP1CDKN1A and CDKN1B organize CDK inhibition. These experiments also demonstrated that FOXP2 induces CDKN1A / p21 expression in osteoblasts and serves as a primary trigger for cell growth arrest. FOXP2 reduction resulted in strong CDKN1A / p21 and slight CDK4 decline and showed no effect on p53 level. Increased p53 / p21 level had no effect on FOXP-2 level. Immunoprecipitation did not show direct FOXP2 / p21 binding sites and it was demonstrated that although increased FOXP2 expression was accompanied by concomitant IL-6 expression but FOXP2 did not controls p21 expression via IL-6, p21 induction by the FOXP2 takes place indirectly. This is still to be studied. The researchers found a correlation between Lhx8 deficiency factor and FOXP2 expression. There may be a relationship between the FOXP2 and the Soxp2.

      Yan X et al. showed 2015 in „Downregulation of FOXP2 promoter human hepatocellular carcinoma cell invasion“ that FOXP2 level in human hepatocellular carcinoma cells is significantly reduced compared to adjacent non-tumor tissue. They used Western blot, transwell assays and immunohistochemistry to measure FOXP2 expression in HCC and in adjacent normal tissues of 50 patients. Their results showed that FOXP2 expression was down-regulated in HCC tumor tissue and that FOXP2-level decrease correlated with poor survival rates. FOXP2 could decrease cell invasion and affect the expression of vimentin and OXE-cadherin.

       Since the study by Schroeder and Myers (2008) has emphasized the importance of cell-specific FoxP expression it is especially important to measure it in different tissues and to take a closer look at its cell-specific interaction mechanisms. Chiu et al. (2014) described how FOXP2 inhibits the ERK / MAPK (HSPB7), NOS1, KCNJ15 and SHH pathways (PTCH1) and Vernes et al. (2011) and Ayub et al. (2013) how it influences the oncogene CDK8. FOXP2 effects Bcl-2 expression which seems to be involved in pro-caspase activation and apoptosis. (Devanna et al., 2014). Spiteri et al. (2007) described the cancer-related actin-binding protein genes TAGLN, NOS1, LBR, KCNJ15 and ANK1 as additional FOXP2 target genes. FP2 affects the proto-oncogene C-MET by binding its regulator the AP1-NFAT complex. It also affects the p53 which in turn binds to C-MMET. (Mukamel et al., 2011) (Ho and Crabtree, 2006). MAPK and TGF-β pathways are modified by lncPINT, which is also affected by the with PRC2 interacting p53.

      FOXP1 functions, parallels to FOXP2 Like FOXP2's, FOXP1 plays an important role in brain development, because its haploinsufficiency is associated with speech deficits and autism. (Tamura et al., 2003; Le Fevre et al., 2013; Horn et al., 2010; Hamdan et al., 2010; Bacon and Rappold, 2011; Lien et al., 2001; Pariani et al, 2009; Chien et al., 2013; Palmerbo et al., 2013; Carr et al, 2010; Vargha-Khadem et al, 1998; Watkins et al. Brain, 2002; Reimers-Kipping et al. al., 2011; Scharff et Petri, 2011; Li et al., 2015; Song et al., 2015) However, according to „Equivalent missense variant in the FOXP2 and FOXP1 transcription factors causes distinct neurodevelopmental disorders“ (2017) of Sollis et al. the R514H mutation of the FOXP1 gene has a wider and heavier disease manifestation than corresponding p.R553H mutation of the FOXP2 gene. Other functions of the FOXP1 gene were described by Rousso et al. (2016), Wang et al. (2003, 2004), Xing et al.(2017) and Taskapilioglu et al. (2016). According to the recent study by Fröhlich et al. „Foxp1 expression is essential for sex-specific murine neonatal ultrasonic vocalization“ (2017), Foxp1 expression is essential for sex-specific murine neonatal ultrasound vocalization. This study could be of clinical importance because boys are more affected by utism and other speech and speech deficits than girls. They showed that Foxp1 and the androgen receptor are co-expressed in striatal neurons and that brain-specific androgen receptor KO (ArNesCre)