Compartmentalization and Interaction of Pax5 and Pax6 in Brain of Mice

Many transcription factors play important roles to maintain the microenvironment, integrity of the blood-brain barrier, the neurons-glia interaction, activities of microglia, composition of cerebrospinal uid, metabolic activities, concentration of neurotransmitters, presence of inammatory and anti-inammatory cytokines, ischemia, stress, aging, neurological disorders, and diseases. The Paired box transcription factors and multifunctional proteins, Pax6 and Pax5 are expressed in brain. They regulate several regulators from cell cycle to cell death. The Pax5, a B-cell lineage-specic activator protein (BSAP), is expressed in the cerebellum, cerebral cortex, hippocampus, olfactory bulb, third ventricles, and choroid plexus. The Pax5 has been observed down-regulated in autism, mental retardation, and Glioblastoma multiforme. The Pax6 affects genes of neurodegeneration, immunological surveillance, and energy homeostasis in brain of mice. The Pax5 and Pax6 recognize several similar DNA sequences and regulate the expression of genes in a tissue-specic manner. Therefore, it is presumed that Pax5 and Pax6, are compartmentalized in brain of mice. Results indicate interactions, cell and tissue-specic compartmentalization, and co-localization of Pax5 and Pax6 in the cerebral cortex, cerebellum, and hippocampus in brain of mice.


Introduction
The Paired box family transcription factors prove critical for development, growth, differentiation, organogenesis, and maintaining functional anatomy of vital organs like eye, nose, limb, muscles, kidney, vertebral column, and brain [1][2][3]. During development, paired box genes regulate cell fate speci cation, proliferation, and/or migration of neuroectodermal precursor cells. The overexpression of Pax genes appears to elicit the transformation of rat broblasts in vitro [4][5][6]. Among them, Pax5 and Pax6 have mainly been described as critical for the development and maintaining functional anatomy of the brain.
The Pax5 has been implicated in B-cell malignancies, dedifferentiation to uncommitted progenitors in the bone marrow, and BCR signaling. The targeting of Pax5 in mice causes defects in midbrain, abolition of lymphopoiesis, and incomplete V-H gene recombination in the adult bone marrow. The impact of Pax5 has been observed on neuronal development, non-hematological astrocytomas, medulloblastoma, and neuroblastomas malignancies. The Pax5 also regulates NF-κB, genes involved in actin-remodeling [7], and the immune pathway in the development of Glioblastoma multiforme [8]. Recent reports suggest multiple binding sites for Pax5 in the promoter of Ionized calcium-binding adapter molecule 1 (Iba1), which regulates actin-bundling, membrane ru ing, cell migration, and phagocytosis in activated microglia. The Pax5 interacts with proteins essential for neuronal and glial differentiation. The interaction of Pax5 with Iba1 indicates Pax5-mediated transcriptional regulation of Iba1 in brain and impact on microglia-mediated immunity in brain of mice [9].
The Pax6 regulates astroglial de-differentiation, neural and glial proliferation in the central nervous system [10][11]. The expression of astrocytes markers GFAP and S100β have been proposed to be regulated by Pax6 as their expression was observed downregulated in Pax6 knockdown condition [12].
The Pax6 binds to promoter sequence elements of Gfap, S100β, Tmem119 [13] and interacts with Iba1 both at the genetic and protein level [14].
The consensus recognition sequence of the Pax6 paired domain deviates primarily only at one position from that of Pax5, and the two proteins exhibit largely different binding speci cities for genes [15] in a tissue-dependent manner. Since, both Pax5 and Pax6 are expressed in the brain, it is presumed that they may have similar DNA binding a nity on genes being expressed in that regions. The information about association of Pax5 and Pax6 in brain is lacking. They may co-localize and physically interact apart from their distinct expression pattern. Observations favor compartmentalization, co-localization, and interactions of Pax5 and Pax6 in brain of mice.
Material And Methods

Animal model
The male adult albino mice of AKR strain were used for the experiments. Mice were maintained at 25 ± 2°C as per the guideline of the Institutional Animal Ethical Committee in the animal house of the department. The mice were sacri ced by euthanasia and cervical dislocation to dissect out brain for experimental purposes. The experiments were carried out three times using brain of adult male mice (n = 5) per experiment.
In silico analysis of interacting proteins and promoter sequence elements of Pax5 and Pax6 The Pax5 and Pax6 interacting proteins in mice were analyzed using http://string-db.org/ and evaluated by interaction scores shown in the string database. Annotation of Pax5 and Pax6 biological functions and signaling pathways were based on the interacting proteins, separately. The promoter sequences of Mus musculus Pax5 and Pax6 were retrieved from Eukaryotic Promoter Database (https://epd.vitalit.ch/index.php). The transcription factor search motifs for binding of Pax5 on Pax6 promoter sequence and Pax6 on Pax5 promoter sequences were analyzed.
Chromatin-Immunoprecipitation (ChIP) with anti-Pax5 and anti-Pax6 in brain of mice The Chromatin Immunoprecipitation was performed as described earlier [13]. Brie y, the lysate of the adult brain was prepared. Cross-linking and chromatin preparation from lysate was done by 1% formaldehyde. The cross-linking reaction was stopped by adding 125mM glycine. Nuclear extract was collected by centrifugation at 10,000xg for 10 min. Nuclear lysis was performed in ChIP-lysis buffer followed by sonication. Typically four rounds, 30-sec pulse with 1 min rest in between rounds at output 5.0 (LABSONIC L, B. Braun Biotech International GmbH, Germany). The desired DNA fragment was between 0.5kb to 1kb in length. The supernatant after sonication containing chromatin was incubated for and incubation at 65 o C for 1 hour. DNA obtained through Immunoprecipitation was puri ed through the phenol: chloroform puri cation method. The pulled DNA was checked on 1% agarose gel. Chromatin prepared without antibody was reverse linked to obtaining input DNA. The qPCR was performed to calculate fold enrichment of promoter Pax5 and Pax6 gene in input DNA, anti-Pax5 and anti-Pax6 pulled DNA and negative control. The primers sequence used were Pax5F, 5′ CGGACCATCAGGACAGGA 3′, Pax5R, 5′ GGGCTCGTCAAGTTGG 3′; Pax6F, 5′GAGGTCAGGCTTCGCTAATG 3′, Pax6R, 5′ TCCAACAGCCTGTGTTGTTC 3′.
Analysis of the interaction of Pax5 and Pax6 by Co-Immunoprecipitation (Co-IP) in brain of mice For Co-Immunoprecipitation, 50µl of Protein-A bead was taken into a spin column and washed twice with 1ml 1X IP buffer by centrifugation at 3000rpm for 20 seconds. 2µg of anti-Pax5 (anti-mouse, sc-13146, Santa-Cruz Biotech, USA) and anti-Pax6 (anti-mouse, sc-32766, Santa-Cruz Biotech, USA) diluted to 200µl in buffer were added to each column containing resin, respectively. The columns were incubated at 4°C for 30 minutes. After 30 minutes, the resin was washed with 1ml cold 1X IP buffer thrice and 150µl of adult mice brain tissue lysate was added to each column, respectively. Then columns were incubated for 1hr at 4°C and washed with cold 1X IP buffer thrice. In the negative control, the beads were incubated with antibodies and lack brain tissue lysate. After washing, 100µl of sample loading buffer was added to the resin, mixed well and the suspension was transferred into the 1.5ml microfuge tube [14,16]. Samples were heat-denatured for 5 minutes, centrifuged for 1 min at 3000rpm and 50µl of the samples were resolved through 12% SDS-PAGE and analysed by Western blotting for Pax5 and Pax6-reactive peptide band, respectively.
Analysis of expression and co-localization of Pax5 with Pax6 in brain of mice The antigen retrieval of cryo-sections was done in 0.1% trypsin+0.1% CaCl 2 for 10 min. Sections after antigen retrieval were blocked with 1% BSA for 1 hour. For double labeling, anti-Pax5 (anti-mouse, sc-13146, Santa-Cruz Biotech, USA, 1:500 dilution) and anti-Pax6 (anti-mouse, sc-32766, Santa-Cruz Biotech, USA, 1:200 dilution) antibodies were used at 4°C for overnight. The sections were washed with PBS and probed with TRITC (red) goat anti-mouse IgG secondary antibody, FITC (green) conjugated goat antimouse IgG secondary antibody (1:2000 dilution) (Merk, India), separately for 2h each for detecting Pax5 and Pax6 immunoreactivity. In the negative control, slides were stained with TRITC (red) goat anti-mouse IgG secondary antibody, FITC (green) conjugated goat anti-mouse IgG secondary antibody (1:2000 dilution) without incubating with primary antibody. The slides were washed with PBS with Tween 20 (0.02%) and counterstained with DAPI (Molecular Probe) for nuclear staining as previously described [14,16]. Imaging was performed using a uorescence microscope (Evos FLc) and confocal microscope (Zeiss LSM 780). Image analysis was performed by Zen software.

Results
Pax5 and Pax6 show interacts and is involved in various signaling pathways Data curation for interacting proteins (Figure 1) and functional enrichment analysis (Supplementary   Table 1 Figure 6C). In the cerebellum, localization of Pax5 and Pax6 was observed throughout the cell body of Purkinje cell. Apart from Purkinje cells, localization of Pax5 and Pax6 were observed in basket cells, stellate cells of the molecular layer, and granule cells respectively show differential co-localization ( Figure 6A). In the cerebral cortex, localization of Pax5 and Pax6 compartmentalized and shows co-localization at some regions on the periphery of pyramidal cells and glial cells whereas stellate cells were observed as complete positive for Pax5 and Pax6 ( Figure 6B). In the hippocampus, localization of Pax5 and Pax6 were observed in basket cells (BC), granule cells (AGC), and radial glia cells ( Figure 6C).

Discussion
Analysis of signaling pathways of Pax5 and Pax6 based on interacting proteins neural network indicates their involvement in transcriptional misregulation and opposite role of Pax5 and Pax6 in cancer malignancies. The misappropriate expression of Pax5 leads to malignancies of gliomas and medulloblastomas which correlates positively with cell proliferation and inversely with neuronal differentiation [4,17] whereas Pax6 act as a glioma tumor suppressor [18][19]. The involvement of Pax5 and Pax6 in TGF-β signalling pathway indicates the association of Pax5 and Pax6 in neuroprotective and immune modulator functions [20]. It also correlates with Huntington's disease pathway and modulations in DNA methylation pattern of Pax6 [21] and the association of Pax5 expression with clinical covariates of disease [22]. Pax6 acts as a downstream target of the Wnt/ß-catenin pathway, and ß-catenin/Pax6 signalling which is critical for self-renewal and neurogenesis of radial glia/neural stem cells during neocortical development [23]. However, Pax5 functions as a transcription factor in canonical Wnt signalling which determines cellular fate in the cerebral cortex and hippocampus [24][25]. Conditional knockout of Pax5 in GABAergic neurons [25] in mice indicates the necessity of Pax5 in normal ventricular development. It has also been observed down-regulated in bipolar disorders and autism spectrum disorder [26].
Results of Chromatin Immunoprecipitation (ChIP) support binding of Pax5 on the promoter sequences of Pax6 and the Pax6 on the control region of Pax5. It also suggests transcriptional regulation of Pax5 and Pax6 by each other, respectively. The observation on the physical interaction of Pax5a/b and Pax6 may be a cooperative binding or complex for other regulations of pathways. The physical association of Pax5 and Pax6 may also in uence their own expression. The pattern of cell-speci c expression and colocalization of Pax5 and Pax6 in cerebellum, cerebral cortex, and hippocampus indicates their compartmentalization in brain of mice. The differential co-localization of Pax5 and Pax6 were observed in parallel bre, stellate cells, glial cells of the molecular layer and granule cells of the granular layer in cerebellum. The expression of Pax6 and Pax5 in granule cells may be associated with ataxia, learning and memory [27]. In the cerebral cortex, the glial cells showed differential localization of Pax5 and Pax6.
The Pax6 compartmentalize in some regions at the periphery where it co-localized with Pax5, which has higher expression at some regions in the periphery and basal expression throughout the cell in pyramidal cells whereas stellate cells showed co-localization of Pax5 and Pax6. Pyramidal cells are the building blocks for high-level functions like memory and consciousness and are thought of as the mover and shaker of the brain [28] correlate with signi cant electrophysiologic changes and cognitive decline with age [29][30]. In the hippocampus, the co-localization of Pax5 and Pax6 were observed in the granule cells and basket cells of dentate gyrus except for a cell that showed Pax6 expression throughout the cell and a very low signal of Pax5 in the middle of the cell. Granule cells signi cantly contribute to learning and memory, which decrease with aging [27]. Adult-born granule cells (aGCs) shown positive for Pax5 and Pax6 have been implicated in cognition and mood actively participate in the encoding of novel information [31]. The heterogeneous population of cells in brain showed cells positive for Pax5 and Pax6, respectively. They may have an independent function and cells positive for both Pax5 and Pax6 may have co-operative functions too in the brain.

Conclusion
Results indicate compartmentalization and co-localization of Pax5 and Pax6 in the cerebral cortex, cerebellum, and hippocampus in brain of mice. Pax5 and Pax6 also interact physically in brain of mice both at genetic and protein levels. Thus, apart from independent roles, Pax5 and Pax6 co-operate to maintain the functional anatomy of the brain (Figure 7). Figure 1 In silico analysis showed a neural network of top 100 Pax5 (A) and Pax6 (B) interacting proteins     In silico analysis of binding of Pax5 and Pax6 to the genetic sequence element of promoter sequence element of Pax6 and Pax5. Evaluation of enrichment of Pax6 and Pax5 promoter sequence in ChIP DNA with anti-Pax5 (A) and anti-Pax6 (B) and IgG pulled DNA (-ve control) in brain of mice. Data are represented as mean ± SEM and different superscripts denote signi cant difference (p≤0.05) in respect to control (independent-samples t-test).

Supplementary Files
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