Alternative splicing of medaka bcl6aa and its repression by Prdm1a and Prdm1b

TL;DR: Xiaomei Ke Central China Normal University Runshuai Zhang CentralChina Normal University Qiting Yao Central ChinaNormal University Shi Duan Central Chinanormal University Wentao Hong Central China normal university Mengxi Cao Jianghan University Qingchun Zhou Central China National Normal University Xueping Zhong Central China NTU School of Life Sciences.

Abstract: Bcl6 and Prdm1 (Blimp1) are a pair of transcriptional factors that repressing each other in mammals Prdm1 represses the expression of bcl6 by binding a cis-element of the bcl6 gene in mammals The homologs of Bcl6 and Prdm1 have been identified in teleost fish However, whether these two factors regulate each other in the same way in fish like that in mammals is not clear In this study, the regulation of bcl6aa by Prdm1 was investigated in medaka The mRNA of bcl6aa has three variants (bcl6aaX1-X3) at the 5'-end by alternative splicing detected by RT-PCR The three variants can be detected in adult tissues and developing embryos of medaka Prdm1a and prdm1b are expressed in the tissues and embryos where and when bcl6aa is expressed The expression of prdm1a was high while the expression of bcl6aa was low, and vice versa, detected in the spleen after stimulation with LPS or polyI:C In vitro reporter assay indicated that bcl6aa could be directly repressed by both Prdm1a and Prdm1b in a dosage-dependent manner After mutation of the key base, G, of all predicted binding sites in the core promoter region of bcl6aa, the repression by Prdm1a and/or Prdm1b disappeared The binding site of Prdm1 in the bcl6aa gene is GAAAA(T/G) These results indicate that both Prdm1a and Prdm1b directly repress the expression of bcl6aa by binding their binding sites where the 5'-G is critical in medaka fish

Summary

Possible Mechanisms of Oxygen Exchange for Dual Phase Composites

• Oxygen exchange on dual-phase composites have been discussed by several groups as mentioned in the introduction.
• Different involvements of the two phases in the oxygen exchange were proposed.
• S1 is the surface area of the P1 phase, V1 is the volume of P1, t is time and k1 is the efficient surface exchange coefficient of the P1 phase.
• The kex-LSF (GB) and kex-CGO (GB) are the effective surface exchange coefficient on the single-phase grain boundaries and kex(TPB) is the effective surface exchange coefficient on the dual-phase grain boundaries (as illustrated in Figure 2II).
• The oxygen exchange on the “cleaned” CGO surface appears to be very fast,40 and possibly, in the older SIMS studies of single phase CGO the exchange reaction has been impeded by unintended impurities on CGO surface.

Experimental

• The sample was sintered at 1300◦C for 10 h to reach 97.4% of the theoretical density, polished and re-annealed at 1000◦C. Characterizations.
• Pt wires attached to both ends of the sample were used as current probes.
• By fitting the conductivity change using Fick’s law of diffusion with appropriate boundary conditions, the values of kex and Dchem can be obtained.

Results

• —The X-ray diffractograms of the pure LSF, the dual-phase LSF-CGO composites after sintering at 1300◦C and of the CGO powder are shown in Figure 3.
• The total conductivity of the composites is lower than that of the pure LSF, due to much lower total conductivity of CGO (Table I).
• At 750◦C the measured values correspond well to the calculated one with τIC of 1 for the CGO70 sample, whereas τIC of 2.5 and 20 were used for the CGO50 and CGO30 samples, respectively (Figure 8B) to match the measured and calculated Dchem values.
• Assuming that oxygen exchange may also occur on the CGO, the effective kex(cal) was calculated according to Eq. 3, where the kex(CGO) was selected as to give a best fit of Eq. 3 to the measured values for different temperatures and pO2’s.

Discussion

• In case of pure LSF the surface exchange reaction at high pO2’s (10−3 < pO2 < 1 atm), by assuming the involvement of oxygen vacancies in the oxygen surface exchange process,10 will be strongly depended on the mobility and/or concentration of oxygen vacancies, while for pure CGO the oxygen exchange will be presumably limited by the electronic conductivity.
• Hence, it could be, that a cleaning of the CGO surface takes place via the presence of LSF, making the oxygen exchange on CGO much faster than when considering single phase CGO samples.
• It should also be pointed out that these two mechanisms are not mutually exclusive and can act simultaneously. ) unless CC.
• Further, the pO2 dependence of kex, which was observed to vary with the CGO fraction and the temperature, can point out different aspects of the mechanism of the oxygen exchange.
• At 750◦C the measured values for the CGO70 sample behaves as expected from the materials properties and increasing the LSF fraction leads to large deviations from the values obtained by volume weighting average, which is interpreted as a tortuosity effect.

Conclusions

• Considering all experimental data it is further suggested that the rds is temperature and composition dependent.
• The involvement of CGO in the oxygen exchange can be through the spillover of oxygen ions, partly reduced oxygen species, from LSF to CGO, where they dissociate and/or incorporate.
• An alternative effect could be that the LSF scavenges impurities from CGO and thereby activates the CGO surface for the oxygen surface exchange reaction.
• Both proposed mechanism could account for the observations.

Figures

Figure 3

Figure 1

Table 2 The predicted N-terminus, half-life, and relative translation e ciency of Bcl6aa by TermiNator

Figure 2

Full text

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Alternative splicing of medaka bcl6aa and its
repression by Prdm1a and Prdm1b
Xiaomei Ke
Central China Normal University
Runshuai Zhang
Central China Normal University
Qiting Yao
Central China Normal University
Shi Duan
Central China Normal University
Wentao Hong
Central China Normal University
Mengxi Cao
Jianghan University
Qingchun Zhou
Central China Normal University
Xueping Zhong
Central China Normal University
Haobin Zhao (  zhaohb@mail.ccnu.edu.cn )
Central China Normal University School of Life Sciences https://orcid.org/0000-0003-4672-7654
Research Article
Keywords: Prdm1/Blimp1, Bcl6, Gene expression, Cis-element, Reporter Assay, Medaka
Posted Date: March 26th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-331464/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

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Abstract
Bcl6 and Prdm1 (Blimp1) are a pair of transcriptional factors that repressing each other in the mammals.
Prdm1 represses the expression of
bcl6
by binding a cis-element of
bcl6
gene in mammals. The
homologs of Bcl6 and Prdm1 have been identied in teleost sh. However, whether these two factors
regulate each other by the same way in sh as that in the mammals is not clear. In this study, the
regulation of
bcl6aa
by Prdm1 was investigated in medaka. The mRNA of
bcl6aa
has three variants
(
bcl6aaX1-X3
) at the 5′-end by alternative splicing with different promoters detected by RT-PCR. The three
variants can be detected in adult tissues and developing embryos of medaka. The predicted proteins of
Bcl6aaX1-X3 may have modication such as acetylation, C-mannosylation, phosphorylation, and
sumoylation in the N-terminuses with different half-lives and relative translation eciencies.
Prdm1a
and
prdm1b
are expressed in the tissues and embryos where and when
bcl6aa
is expressed. The expression
of
prdm1a
was high while the expression of
bcl6aa
was low, and vice versa, detected in the spleen after
stimulation with LPS or polyI:C. In vitro reporter assay indicated that
bcl6aa
could be directly repressed by
both Prdm1a and Prdm1b in a dosage-dependent manner. After mutation of the key base, G, of all
predicted binding sites in the core promoter region of
bcl6aa
, the repression by Prdm1a and/or Prdm1b
disappeared. The consensus binding site of Prdm1 in
bcl6aa
gene is GAAAA(T/G). These results indicate
that both Prdm1a and Prdm1b directly repress the expression of
bcl6aa
by binding the consensus
binding site where the 5′-G is critical in medaka sh.
Introduction
Bcl6 (B-Cell Lymphoma 6) is a member of the POK (POZ and Krüppel) ⁄ZBTB (zing nger and BTB)
protein family (Lee and Maeda 2012). Prdm1 (positive regulatory domain I-binding factor or PR domain-
containing protein 1) also called Blimp1 (B lymphocyte-induced maturation protein 1) is belonging to
PRDM family (John and Garrett-Sinha 2009). Bcl6 and Prdm1 are two transcription factors functioning in
diverse tissues such as the immune system and bone, etc. in the mammals. The effect of Bcl6 is opposite
to that of Prdm1. For example, Bcl6 inhibits but Prdm1 promotes osteoclastogenesis in mice (Miyauchi et
al. 2010). In the immune system, Prdm1 is highly expressed in the Th2 (T helper 2) cells and is required
for normal Th2 humoral responses in vivo by repression of Bcl6 and Tbx21 (T-box transcription factor 21)
which are necessary for Th1 cells (Cimmino et al. 2008). Bcl6 promotes differentiation of CD4 (cluster of
differentiation 4) + T follicular helper (Tfh) cells and B cells in mice. Contrarily, Prdm1 inhibits Tfh
differentiation and B cell maturation (Johnston et al. 2009). STAT3 (Signal transducer and activator of
transcription 3) can upregulate PRDM1 coordinately with down-regulation of BCL6 to control human
plasma cell differentiation (Diehl et al. 2008). Prdm1 and Bcl6 repress one another in CD4 T cells. Bcl6
directly inhibits
prdm1
expression or binds to Bach2 (BTB domain and CNC homolog 2) to repress
prdm1
and represses plasmocytic differentiation (Ochiai et al. 2008; Tunyaplin et al. 2004). Conversely, Prdm1
directly represses
bcl6
by binding to the
bcl6
gene in both CD4 T cells and B cells (Cimmino et al. 2008).
Hobit (Homolog of Blimp1 in T cells) or ZNF (Zinc nger protein) 683 is the homolog of Prdm1 in
mammals. Hobit was initially identied in natural killer T (NKT) cells of mouse (van Gisbergen et al.

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2012). Hobit functions in repression of IFN (Interferon)-γ expression and induces granzyme B expression
in mice (van Gisbergen et al. 2012). Human HOBIT was identied in NK cells and effector-type CD8 + T
cells (Vieira Braga et al. 2015). Hobit recognizes similar regulatory sequences of the target genes of
Prdm1 in mouse lymphocyte (Mackay et al. 2016). Hobit cooperates with Prdm1 in differentiation and
maintenance of CD4 + or CD8 + tissue-resident memory T (Trm) cells (Behr et al. 2019; Kragten et al. 2018;
Mackay et al. 2016; Zundler et al. 2019).
In teleost sh, the homologs of Prdm1 are Prdm1a, Prdm1b, and Prdm1c. Prdm1a and Prdm1b are
closely related to mammalian Prdm1 and Hobit respectively. Prdm1c is evolved from duplication of
Prdm1a in sh (Perdiguero et al. 2020). Prdm1a has been reported in fugu (Ohtani and Miyadai 2011;
Ohtani et al. 2006a), zebrash (Ingham and Kim 2005; Page et al. 2013; Roy and Ng. 2004; Wilm and
Solnica-Krezel 2005), rainbow trout (Diaz-Rosales et al. 2009; Perdiguero et al. 2020; Zwollo 2011),
medaka (Zhao et al. 2014), Nile tilapia (Wu et al. 2019), and Japanese ounder (Liu et al. 2016). Prdm1a
plays important roles in embryonic development such as n, muscle, and cloaca, etc. of zebrash
(Ingham and Kim 2005; Mercader et al. 2006; Pyati et al. 2006; Roy and Ng 2004; Wilm and Solnica-Krezel
2005). Prdm1a was detected in IgM + CD8α- cells in fugu kidney (Odaka et al. 2011). Prdm1a is
expressed in the plasma CD45 + B cells with expression of IgM in zebrash (Page et al. 2013). Previously,
we reported the expression of
prdm1a
(ENSORLG00000015684, JX402912) and
prdm1c
(ENSORLG00000012948, JX402913, NP_001265739) in medaka (Zhao et al. 2014).
Prdm1a
could be
upregulated in the liver of medaka and zebrash by lipopolysaccharide (LPS), polyinosinic:polycytidylic
acid (polyI:C), and grass carp reovirus (GCRV) (Zhao et al. 2014). Prdm1 was also detected in the IgM + B
cells of the head kidney of tilapia and was stimulated with LPS in vitro (Wu et al. 2019). Rainbow trout
prdm1a
could be upregulated by IL-2 (Diaz-Rosales et al. 2009). Rainbow trout
prdm1a
,
prdm1b
, and
prdm1c
transcripts were identied in the B and T cells and were upregulated in the head kidney and
spleen after infection of Viral hemorrhagic septicemia virus (VHSV) (Perdiguero et al. 2020).
The homologs of Bcl6 have been reported in several sh including fugu (Odaka et al. 2011; Ohtani et al.
2006b), zebrash (Lee et al. 2013), medaka (Zhang et al. 2019b), grass carp (Zhu et al. 2019), and
Senegalese sole (
Solea senegalensis
) (Ponce et al. 2020). Zebrash Bcl6a is required for optic cup
formation (Lee et al. 2013) and is a key factor for cold response (Hu et al. 2015). Fugu Bcl6 was identied
in the immune organs or tissues (Ohtani et al. 2006b), and in the leukocyte cells expressing secretory-type
IgM and Prdm1 (Odaka et al. 2011). Transcription of bcl6 could be promoted by T-cell factor (TCF) 7 in
response to GCRV challenge in grass carp (Zhu et al. 2019). Senegalese sole
bcl6
could be induced by the
sulfated polysaccharide ulvan from a green seaweed (
Ulva ohnoi
) (Ponce et al., 2020). Previously, we
reported that two homologs of
bcl6
,
bcl6aa
and
bcl6ab
, were detected in the immune organs such as the
liver, kidney, and spleen, and could be induced by polyI:C and LPS in medaka (Zhang et al. 2019b).
The reports mentioned above show the conserved function of Bcl6 and Prdm1 in the immune response in
sh. Fugu Bcl6aa and Prdm1a were reported as the transcriptional repressors in vitro (Ohtani and
Miyadai 2011). A mutation of possible Bcl6 binding site in the 5′-regulation region of
prdm1
gene of
Japanese ounder increased the reporter activity in vitro (Li et al. 2017). The possible binding sites were

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also found in rainbow trout
prdm1
genes (Perdiguero et al. 2020). However, whether Prdm1 and Bcl6
repress each other in the same way as that in mammals is not reported. Although the expression of
prdm1a
,
prdm1c
, and
bcl6aa
had been reported previously (Zhang et al. 2019b; Zhao et al. 2014), the
expression of
prdm1b
and the alternative splicing variants of
bcl6aa
are not reported yet in medaka. In
this paper, we report the alternative splicing variants of
bcl6aa
and the expression of
prdm1b
in medaka.
Moreover, the repression of medaka
bcl6aa
by Prdm1a and Prdm1b was studied in vitro. The results
showed a direct repression of
bcl6aa
by medaka Prdm1a and Prdm1b binding the conserved cis-
elements.
Materials And Methods
Animals
Wild strain medaka was used as experimental sh. The sh were maintained under articial photoperiod
of 14 h light and 10 h dark, at an ambient temperature of 28.0°C. Spontaneously spawned eggs were
collected and incubated at an ambient temperature of 28.0°C.
Adult sh were randomly divided into three groups and were injected intraperitoneally with 10 µl of
phosphate buffer solution (PBS), LPS (Sero-type: O55:B5, Sigma-Aldrich, Merck KGaA, Darmstadt,
Germany), polyI:C (Sigma-Aldrich), respectively (Zhao et al. 2014; Zhang et al. 2019). LPS and polyI:C
were dissolved in PBS in a concentration of 5 µg/µl respectively. Five sh of each group were randomly
sampled at 1–10 days post injection (dpi) for measurement of gene expression.
The animal protocol for this study was approved by the Animal Care and Use Committee of Hubei
Province in China [No.
This study was carried out in strict accordance with recommendations in the Regulation for the
Management of Laboratory Animals of the Ministry of Science and Technology of China.
The animal protocol for this study was approved by the Animal Care and Use Committee of Hubei
Province in China [No.
This study was carried out in strict accordance with recommendations in the Regulation for the
Management of Laboratory Animals of the Ministry of Science and Technology of China.
This study was carried out in strict accordance with recommendations in the Regulation for the
Management of Laboratory Animals of the Ministry of Science and Technology of China. The animal
protocol for this study was approved by the Animal Care and Use Committee of Hubei Province in China
[No. SYXK(E)2015-0012].
Extraction of total RNA
The cDNA was synthesized according to the protocol of FastQuant RT kit (Tiangen Biotech, Beijing,
China).
Adult tissues were isolated from medaka sh that were killed by decapitation after anesthesia with MS-
222 (Sigma-Aldrich). Total RNA from adult tissues and embryos were extracted by Ultrapure RNA kit

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(CoWin Biosciences, Beijing, China) as the protocol provided by the manufacturer. The cDNA was
synthesized according to the protocol of FastQuant RT kit (Tiangen Biotech, Beijing, China).
Detection of
bcl6aa
,
prdm1a
, and
prdm1b
in adult tissues and embryos by RT-PCR
There are two transcripts
bcl6aa-201
(ENSORLT00000030671.1) and
bcl6aa-202
(ENSORLT00000019521.2) of medaka
bcl6aa
(ENSORLG00000015589) predicted in the Ensembl
(http://www.ensembl.org/Oryzias_latipes/). To conrm the prediction, we detected these two transcripts
by RT-PCR using the primers designed on the genomic sequence of medaka
bcl6aa
(Fig.1A, Table1). The
PCR results were puried and were ligated into pMD18-T vector (Takara Bio, Dalian, China). Positive
colonies of
E coli
. transformed with the ligated vectors were sent for sequencing. Sequencing results were
assembled respectively for each colony and were aligned together. Then, the variants were identied and
were blasted against the genome of medaka on web (http://www.ensembl.org).
The cycling program was 95 ℃ 2 min followed by 39 cycles of 95 ℃ 10 s, 62 ℃ 30 s and 65 ℃ 30 s.
Relative expression of the genes in the samples was calibrated/normalized against RPS18 by using
2−ΔΔCt method (The quantity in the samples of sh received phosphate buffer solution (PBS) referred as
1) (Livak and Schmittgen 2001).
PCR reaction was performed in a volume of 25 µl, containing 12.5 µl of 2× Es Taq master mix (CoWin), 1
µl of primers (10 µmol/L), 0.1 µl of cDNA solution, and 10.4 µl of double distilled water (ddH
2
O). The
cycling program was 95 ℃ 3 min; 30 cycles of 95 ℃ 30 s, 62 ℃ 30 s, 72 ℃ 25 s; and 72 ℃ 5 min.
Quantitative RT-PCR (qRT-PCR) of triplicate samples was performed with CFX96 real-time PCR detection
system (BioRad Laboratories, Hercules, California, USA) in a volume of 20 µl containing template cDNA,
primers and 2× SuperReal Pre Mix Plus kit (Tiangen). The cycling program was 95 ℃ 2 min followed by
39 cycles of 95 ℃ 10 s, 62 ℃ 30 s and 65 ℃ 30 s. Relative expression of the genes in the samples was
calibrated/normalized against
RPS18
by using 2
−ΔΔCt
method (The quantity in the samples of sh
received phosphate buffer solution (PBS) referred as 1) (Livak and Schmittgen 2001). The primers used
were shown in Table1.
Beta-actin
and/or the ribosomal protein
RPS18
were used as internal control
(Zhao et al. 2014).
Bioinformatic assay
The variants of
bcl6aa
were translated into protein isoforms, Bcl6aaX1-X3. The isoforms were analyzed
by SignalP 5.0 Server (http://www.cbs.dtu.dk/services/SignalP/) to nd the signal peptide. The possible
sites of acetylation, C-mannosylation, phosphorylation, and sumoylation of the isoforms were predicted
by the Servers of NetAcet 1.0 (http://www.cbs.dtu.dk/services/NetAcet/), NetCGlyc 1.0
(http://www.cbs.dtu.dk/services/NetCGlyc/), NetPhorest (http://www.netphorest.info/), and SUMOplot
analysis program (https://www.abcepta.com/sumoplot). The N-terminus, protein half-life, and relative
translation eciency of Bcl6aaX1-X3 were predicted by TermiNator (https://bioweb.i2bc.paris-
saclay.fr/terminator3/).
Reporter assay

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Xiaomei Ke Central China Normal University Runshuai Zhang Central China National University Qiting Yao Central China NTU Shi Duan Central China NNU Shi Wentao Hong Central China NCU Mengxi Cao Jianghan University Qingchun Zhou Central China NORU Xueping Zhong Central China normal University Haobin Zhao (  zhaohb @ mail.ccnu.edu.cn ) Central China NHU School of Life Sciences https: //orcid.org/0000-0003-4672-7654