Kitabı oku: «Influence of FOX genes on aging and aging-associated diseases», sayfa 4

Structure and function of FoxP genes is responsible for his function

Structure of the FoxP2 gene and its isoforms

The FOXP2 gene is located on chromosome 7 and contains at least 280 (according to some data 603) kb, many introns (about 280,000 non-coding base pairs, according to a publication in 2007 - 603,000 base pairs), 7 exons ( 2145 coding base pairs), but their number is variable. (Zhang et al., 2002;Wright and Hastie, 2007)

The protein product of the FOXP2 gene consists of 715 amino acids and has the following domains: a highly conserved DNA-binding domain, a 508 to 584 amino acid "winged" helix domain (BHT) and the forkhead box with highly conserved two beta-sheets, three alpha helixes and a helix-turn-helix-motif-wing.

Structural variations occur between the second and third helix. The polyglutamine-rich regions with the repetitive CAG and CAA sequences show a high mutation rate as well as different length in different taxa. The FOXP2 gene has a zip finger involved in protein-protein interactions and a leucine zipper. The DNA binding in the minor and the major groove to various targets occurs between the third alpha helix (recognition helix) and the second wing of the FOX transcript. (Enard et al., 2002;Kaestner et al., 2000; MacDermot et al., 2005) The hinge loop plays the most important role in the FOXP2 protein binding to the target genes and the mutation P539A changes its form.(Morris et al., 2018)

Alternative splicing creates different FOXP2 isoforms and causes changes in FOXP2 activity. (Castellano and Downward, 2011). Depending on the tissue and cell type, FOXP2 expression can be started on at least 4 starting points (TSSs). (Bruce and Margolis, 2002; Schroeder and Myers, 2008).

Regulation of various genes by dimer formation with FOXP genes

FOXP2 cooperation and FOXP homo- and heterodimers A strong cooperation between FOXP members could be due to the fact that the FOXP2, FOXP1, FOXP3, and FOXP4 are 55-65% identical. A possible explanation for this similarity is proposed by Song et al. in "Genesis of the vertebrate FoxP subfamily member genesis during two ancestral whole genome duplication events" (2016). According to Murugan et al. (2013) decreased FoxP2 expression in the striatal region of adult zebra finches also interferes with their sensitivity to dopaminergic regulation in signalling via D1 receptors in Area-X, they also have co-localized dopamine D1A, D1B and D2 receptors in striatal Foxp2-expressing neurons. Dopamine is considered to be an important neurotransmitter whose deficiency causes some neurodegenerative diseases, e.g. aging relevant Parkinson's disease. This disease is characterized among other things by a washed-out language. It would be interesting to investigate if FOXP2 plays a role in these processes. The FoxP proteins can regulate their target genes in various cellular contexts depending on binding cofactor. Different protein combinations can lead to opposite effects. This could explain why certain tissues might be much more susceptible to the effects of mutations than other tissues. This study opened new perspectives in the regulation of FOXP2 target genes via protein-protein interactions between the FoxP family members, enabling a deeper understanding of the combinatorial control between the FoxP2 and its interaction partners. This study provided new basic knowledge not only about birdsong, but also about the neural function of human speech.

FOXP2 regulates the expression of many genes during embryonic development as well as the WNT and Notch signalling pathways. Further interactions have been observed with some histone family members (H2AFX; H3f3B) and two heat shock proteins (Hsp25; Hsp90a).

Importance of FOXP1/2/4 interaction for oncological processes Several FOXP genes have been observed in many aging relevant oncological processes. Foxp1 / 2/4-NuRD interaction is processed by the p66beta, a transcriptional repressor and a component of NuRD. During this process, the chromatin-remodelling complex regulates gene expression. He also influences the Interleukin-6. Interleukin-6 in turn contributes to the epithelial injury response and activates „myeloid cells“. The „myeloid cells“ are generally associated with cancer and stimulate eg. intestinal cells to divide, which leads to colorectal carcinoma. Artavanis-Tsakonas et al. (1999) studied NCOR2 and SNW1 as part of notch-mediated signalling and its role in proliferation and differentiation processes as well as in apoptosis. The NCOR2 is not only a FOXP2 downstream target but also shows interaction with FOXP1 during myocardial development. (Jepsen et al., 2008), (Wilke et al., 2012) NCOR2-mediated regulation can be considered as a common mechanism by which FOXP1 and other members of the FOXP family regulate gene expression during organogenesis. This study showed different effects of six FOXP1 / 2/4 protein combinations on the NCOR2. The FOXP1 / 2 combination showed the strongest effect. All combinations except the FOXP1 / 4 dimer led to increased NCOR2 expression. The FOXP2 homodimers induced decreased SNW1 expression while the FOXP1 and FOXP4 homodimers led to increased expression. The influence of FOXP1 / 2 and FOXP4 / 2 on SNW1 expression seems to be unlikely. These results gave an interesting insight into the dual FOXP2 function both as a repressor and as an activator. This ability for build different dimer-combinations may be a hint to fine-tune cell-specific transcriptional regulation. The FOXP1 / 2/4 dimer combinations are preferred. These results suggest that relative levels of FOXP1 / 2/4 proteins determine FOXP2's ability to act as an activator or repressor. The researchers found also that FOXP1 / 2, FOXP1 / 4 and FOXP2 / 4 are co-localized.

FOXP2 modulation by alternative splicing The FOXP is also regulated by alternative splicing. This way the FoxP2 gets different isoforms and is homodimerized and this leads to a change of its activity. (Santos et al., 2011) Similar results were reported by Chen et al. (2014) and Olias et al. (2013). This FOXP2 modification was observed in the lower and dorsal thalamus, in the striatum (except Area X) and in the cerebellar Purkinje cells. These are brain areas where the FoxP2 is permanent strongly expressed. (Takahashi et al., 2003), (Ferland et al., 2003), (Haesler et al., 2004) Epigenetic FOXP2 Regulation by Methylation FOXP2 methylation plays an important role by adipositas. Spaeth et al. showed in „The FOXP1, FOXP2 and FOXP4 transcription factors are required for islet alpha cell proliferation and function in mice“ (2015) that Foxp2 is important for the growth and function of pancreatic alfa islands. The islets of Langerhans of the exocrine pancreas contain five different cell populations: the beta, the alpha, the delta, the epsilon and the pancreatic polypeptide cells. These secrete the ghrelin and pancreatic polypeptide hormones (insulin, glucagon, somatostatin). The authors noted that interaction between FOXP2 and FOXP1 / FOXP4 is required for alpha-islet cell proliferation of the mice. Adult beta cells normally secrete insulin, the alpha cells - the glucagon. Autoimmune beta-cell destruction causes type 1 diabetes while type 2 diabetes is characterized by insulin resistance in the peripheral tissues. Type 2 diabetes is accompanied by insulin deficiency and the loss of beta cell identity. The transcription factors accomplish the reprogramming of terminally differentiated cells and embryonic stem cells into the beta-like cells. The members of the FOX superfamily play crucial roles in these processes, which is also aging relevant. E.g., FOXA2 is known as a pancreatic cell fate regulator. FOXM1 controls expression of cycle factors and increases beta cell mass during metabolic stress, including pregnancy stress and partial pancreatectomy. Metformin, Berberine, EGCG, quercetin and other natural products activate cancer relevant AMPK and decrease mTORC1 activity. MTORC1 activity is frequently elevated in CSC including pancreatic CSCs (Ming et al., 2014; Rozengurt et al., 2014; Matsubara et al., 2013) EGCG also positively effects cancer and aging relevant p21Cip1 and negatively the PI3K/PTEN/Akt/mTORC1 pathway (Chen et al., 2012)

According to Alessandro et al., (2015) MET positively influences decreased mitochondrial transmembrane potential, sensitivity to TRAIL via DR5-upregulation, ROS-level and cell cycle in cancer via miR-221 (Tanaka et al., 2015 Matsubara et al., 2013) but also via TRAIL interaction. In this case G-Phase arrest happens via p27Kip-1 and via interaction between caspases (Coleman et al., 2013) Other plant-derived cancer relevant chemicals are e.g. SHH pathway regulating catechins cyclopamine, norcantharidin, sulforaphane and zerumbone (Huang et al., 2013) They act best in combination and suppress ALDH1, MMP-2 and MMP-9 via KRAS under- and let7 miR- upregulation. (Appari et al., 2014) Momordica charantia has positive effect on inflammation and on cancer (Dandawate et al.,2016), e.g. on ovarian cancer via of AMPK up-, mTOR/p70S6K and AKT/ERK/FOXM1 signalling cascade underregulation (Yung et al., 2016) possibly via Alpha-Momorcharin (Deng et al., 2014).

Also Rooperol influences apoptosis with the help of mitochondrial membrane potential. It upregulates ROS via TP53 activation, but also OCT4 and stemness relevant SOX2 and NANOG (Ali et al., 2015) It also increases Pomiferin level, which influences BIM1 and NESTIN (Zhao et al., 2013)

FOXP1, FOXP2 and FOXP4 are of great importance for alpha cell proliferation and function and are expressed in the pancreas and eyes of Xenopus laevis during its development. FOXA1 and FOXA2 also regulate glucagon production and secretion-controlling genes: the MAFB, the Brn4 (also known as the Pou3f4), the PCK2, the Nkx2-2, the Kir6.1 (also known as the Kcnj8), the Sur1 (also known as the ABCC8) and the GIPR.

FOXP2 regulation by external factors Regulation by PH level Blane and Fanucchi (2015) studied in "Effect of pH on the Structure and DNA Binding of the FOXP2 Forkhead Domain." effects of pH from changes 5 to 9 on FOXP2 function and reported that a change in pH (pH 7.5) directly affects the FOXP2 binding affinity via the altered hydrogen bonding pattern. This is due to the protonation or deprotonation of His554 (the amino group of its imidazole side chain, pKa ~ 6.5). The researchers used as methods gel permeation chromatography, ultraviolet circular dichroism, intrinsic and extrinsic fluorescence etc. Their results showed that the pH does not affect the protein secondary structure in the presence or absence of DNA but alters its tertiary structure. The protein showed a less compact structure at low pH in the absence of DNA. When the DNA was added, the protein became more compact, even at low pH and its dimerization potential increased. They regarded the pH as a regulatory mechanism of FOXP2 forkhead domain (FHD) transcription that interacts with the DNA by helix placement in the major groove. These results could also be important in cancer tissue, where FOXP2 expression plays an important role. E.g., not only the genetic component but also a previous chronic gastric ulcer with changed pH plays a significant role in gastric cancer. The gastric ulcer-causing Helicobacter pylori gains an almost pH-neutral environment with the help of the enzyme urease, which splits the urea into carbon dioxide and ammonia. It would be of great scientific interest to investigate whether the change in the gastric pH together with FOXP2 have an effect on the development of gastric cancer.

Regulation through Vitamin-D

Hawes et al. (2015) showed in „Maternal vitamin D deficiency age foetal brain development in the BALB / c mouse“ how the maternal vitamin D deficiency alters foetal brain development in transgenic Balb / c mice. Before and during pregnancy a vitamin D-rich (2.195 IU / kg) or a low (0 IU / kg) diet was given for 5 weeks and the foetal brains were analysed morphologically and for gene expression at 14.5 or 17.5. embryonic day. It was found that the vitamin D deficiency during pregnancy leads to reduction of rump length, lateral ventricle volume and head size. The FoxP2 expression and at the same time the expression of Brain-Derived Neurotrophic Factor (BDNF), the Transforming growth factor-β1 (TGF-β1) and brain tyrosine hydroxylase (TH) in dopaminergic neurons was altered. The vitamin D-poor diet reduced FOXP2 expression in immunoreactive cells and in the developing cortex in female foetuses. These results allowed deeper insight into the medically relevant reasons for foetal degeneration accompanied by prenatal vitamin D deficiency. It is known that vitamin D interacts with neurotrophic factors (NGF, NT3, NT4, GDNF) and its deficiency triggers neurodegenerative diseases in old age. (Jia et al, 2015) It would be of great interest to investigate if this effect is based on the interaction with FOXP2. Since FOXP2 seems to play an important role in many oncological diseases it would be also interesting to investigate if and to what extent this influence is controlled by vitamin D.

FOXP2 regulation by internal factors Regulation by HuR According to Popovitchenko et al. (2016) „Depending on its degree of phosphorylation, the antigen R (HuR) dictates the amount of FOXP2 mRNA and the development of the neocorticals controlled by it, depending on its degree of phosphorylation“ HuR is the main factor in differential translation of autism-associated FoxP subfamily members in the developing neocortex subpopulation of the projection neurons. Regulation by Risperidone and NAP The study of a big Scottish family with severe mental disorders and schizophrenia (Liu et al. 2009) showed a balanced chromosomal translocation [(1:11) (q42.1; q14.3)] and an abnormally-truncated DisC1 protein (a microtubule-regulating and the FOXP2-influenced protein encoded on chromosome 1). The DISC1 is expressed in the cerebral cortex, in the hypothalamus, in the amygdala, in the hippocampal dentate gyrus, in the olfactory bulb and in the cerebellum. The truncated human DISC1 (hDISC1) alters its localization and can no longer interact with microtubules and microtubule-associated proteins. It leads to decreased dendritic growth and branching. Such anomalies have also been found in brain samples from patients with schizophrenia. (Harrison, 2004) This mutation has also been linked to depression and bipolar disorder. (Burdick et al., 2005) A connection between DISC1 and FOXP2 effects human verbal fluency as well as the ability to acquire spoken language. Several studies indicate that FOXP2 polymorphisms are in some cases associated with schizophrenia. (Nicodemus et al., 2014), (Walker et al., 2012), (Lai et al., 2001), (MacDermo et al., 2005), (Sanjuan et al., 2005), (Sanjuan et al. , 2006), (Tolosa et al., 2010) Vaisburd et al. (2015) showed in „Risperidone and NAP protect cognition and normalize gene expression in a schizophrenia mouse model“ that risperidone reduces effects of the DISC1 mutation. Risperidone is an analogue of the microtubule-stabilizing activity-dependent neuroprotective protein (ADNP) with a NAP (NAPVSIPQ) sequence and a SxIP motif (a microtubule junction for microtubule-end binding, responsible for microtubule dynamics and this is also important for synaptic plasticity and neuroprotection). (Oz et al., 2014), (Gozes et al., 2011), (Holtser-Cochav et al., 2006), (Jouroukhin et al., 2013), (Kumar & Wittmann, 2012).

Both risperidone and NAP improved object recognition in the Morris water labyrinth. In contrast to Risperidone, NAP additionally reduced the anxiety in transgenic mice. Doxycycline blocked the expression of the mutated DISC1 gene. The candidate drugs were selected using bioinformatics programs and then affinity chromatographed. Mutations of the DISC1 gene were associated with increased FOXP2 level in hippocampus. FOXP2 levels could be significantly reduced by treatment with NAP, risperidone or their combination. This effect may be due to the blocking of the dopamine and 5HC2A serotonin receptors in the mesolimbic system, which leads to the reduction of negative schizophrenia symptoms. (Meltzer and McGurk, 1999), (Farde et al., 1995)

An increased dopamine level leads to psychotic symptoms. The study by Mendoza et al. (2015), showed that FoxP-expressing neurons in Area-X also contain dopamine receptors 1A, 1B, and 2. Further studies may clarify whether the schizophrenic positive symptoms are due to the dopamine-FOXP2 interaction and how much FOXP 2 can influence dopamine dependent neurodegeneration in aging.

Regulation of FOXP2 and other FOXP genes by SUMOilization

Effect of sumoilization on the FOXP2 gene In „The Key Regulator for Language and Speech Development, FOXP2, is a Novel Substrate for SUMOylation“ Meredith et al. (2016) reported that the FOXP2 is covalently modified on its amino acid residues by both SUMO1 and SUMO3 at phylogenetically conserved lysine 674. The acid residues downstream of the FOXP2 SUMOylation motif are required for full SUMOylation capacity and modulation of its downstream target genes (DISC1, SRPX2 and MiR200c). Sumoylation is a form of posttranslational modification. The SUMOs (small ubiquitin-like modifiers) is a class of regulatory proteins that control the activity of the target protein and its interaction with other proteins. But the modification does not alter the localization and stability of FOXP2. The modulation effect can be reduced by SENP2 (a specific SUMOylation protease). It has also been observed that the human R553H mutation of FOXP2 reduces its SUMOylating ability compared to wild-type FOXP2. Isui et al. (2017) showed in „Sumoylation of FOXP2 Regulated Motor Function and Vocal Communication Through Purkinje Cell“ with the help of in utero electroporation that the SUMOylation of FOXP2 has, among others, an effect on the development of Purkinje cells and plays an important role in motor skills and vocal communication. They identified FOXP2 SUMOylation in the cerebellum of new-born K673 mice with the help of in vivo and in vitro coimmunoprecipitation and concluded that PIAS3 (an E3 ligase of the small ubiquitin-like modifier) catalyses FOXP2 SUMOylation. This SUMOylation modifies the transcription regulation of FOXP2. Estruch et al. (2016) showed in „The language-related transcription factor FOXP2 is post-translationally modified with small ubiquitin-li modifiers“ that post-translational FOXP2 modification is accomplished by members of the PIAS family. These members of the PIAS family are E3 ligases that promote SUMO transfer from the SUMO-conjugating enzyme UBC9 to an acceptor lysine residue of the target protein. (Rytinki et al., 2009) The SUMO2 and the SUMO3 are 95% identical. However, SUMO1 is only to 50% identical to SUMO2 and SUMO3 (Meulmeester and Melchior, 2008). Estruch research group studied various DNA constructs. They used myc-labelled PIAS1 and the His, mCherry and the YFP-labelled SUMOs and the bioluminescence resonance energy transfer assays. For FOXP2 detection they used Western blotting with the anti-V5 antibody and the anti-beta actin as reference. They took UBC9 enzyme to measure the extent of FOXP2 SUMOylation. During the study of FOXP2-PIAS interaction the working group used the BRET assay and the luciferase assays with the Renilla luciferase (Luc). Further methods were the gel-shift assay, the pull-down assay, the yeast two-hybrid assay and the fluorescence microscopy of HEK293 cells. The study revealed that FOXP-2 could be modified by all three human SUMO proteins and that PIAS1 promotes this process. The docking site of SUMO proteins in the FOXP1 and FOXP4 appears to be the most conserved N-terminal region of FOXP2 away from the polyglutamine tract. This region also interacts with the autism-relevant transcription factor TBR1. (Parikshak et al., 2013)