Mechanistic Study The Expression Of Histone Transcription Factor Spt21

In eukaryotes,DNA is compressed in the nucleus in the form of chromatin.As the fundamental structural unit of chromatin,nucleosome is composed of a histone octamer wrapped by 147 bp DNA.Core histones are divided into four types,including histones H2A,H2B,H3 and H4.The expression of core histone is strictly regulated by cell cycle.In the S phase,histone genes are expressed in large quantities.Outside the S phase,the transcription of histone genes is repressed.Strict regulation of histone level is of great significance to cells.Insufficient histone expression will lead to cell cycle arrest,cell dedifferentiation,cell aging and even tumorigenesis.Excessive accumulation of histones will be toxic to cells.Histone expression is regulated by many factors,including inhibitors and activators.Spt21 is a transcription activator for histone genes.Spt21 binds to the promoter region of histone genes through Spt10,and then recruits histone acetyltransferase to acetylate histones in the promoter region,which eventually activates the transcription of histone genes.The expression of Spt21has also been reported to be regulated by the cell cycle.In the S phase,Spt21 protein accumulates in large quantities to activate the transcription of histone genes.Outside the S phase,Spt21 protein is degraded.The expression of Spt21 protein and core histone is coordinated in the cell cycle.Therefore,it is important to study the regulatory mechanism of Spt21 expression in the cell cycle.The degradation pathways of intracellular proteins are mainly divided into ubiquitin-proteasome system and autophagy pathway.In the ubiquitin proteasome degradation system,ubiquitin molecules are covalently attached to protein substrates through three sequential enzymatic reactions.Substrates containing multiple ubiquitin molecules are quickly recruited to the proteasome for degradation.Autophagy pathway is the process of transporting damaged organelles and misfolded or unfolded proteins to lysosomes or vacuoles for degradation.In the G1 phase,APC/C^Cdh1 complex can recognize the KEN region of Spt21and promote the degradation of Spt21 by ubiquitin-proteasome system.However,it remains unknown how Spt21 expression is regulated in other stages of the cell cycle.In this thesis,we mainly studied the regulatory mechanism of Spt21 expression in cell cycle.The research results are as follows:1.The protein of Spt21 is at a low level in cell.Deletion of SPT21 has no apparent effect on cell growth.However,when SPT21 is overexpressed,our growth experiment and gradient dilution assay showed that excessive accumulation of Spt21 protein significantly inhibits cell growth and the ability to resist DNA damage.This indicates that maintaining a normal level of Spt21 is important for cell growth.2.Cell cycle experiments showed that the m RNA and protein levels of Spt21 are strictly regulated by cell cycle.The transcription level of SPT21 peaks in the late G1 phase,and then reduces in other periods.The protein level of Spt21 peaks in the S phase,and reduces in other periods.3.Spt21 can be degraded by autophagy pathway.We knocked out genes involved in proteasome pathway or autophagy pathway,and found that Spt21 protein is degraded by autophagy pathway in S/G2 phase.When cells were grown under rapamycin or nitrogen starvation conditions that induce autophagy,the degradation of Spt21 protein is accelerated.4.In order to identify the autophagy proteins that regulate Spt21 degradation,we examined Spt21 in the deletion mutants of autophagy proteins.The results showed that deletion of ATG7 and ATG8 genes can inhibit the degradation of Spt21 by autophagy.When cells were grown under rapamycin and nitrogen starvation conditions,deletion of ATG7 and ATG8 genes can also inhibit the degradation of Spt21 by autophagy.5.Spt21 can directly interact with autophagy proteins.We performed co-immunoprecipitation assay and found that Spt21 interacts with autophagy proteins Atg7and Atg8.This showed that Atg7 and Atg8 proteins regulate the degradation of Spt21 by autophagy through interaction with Spt21 protein.In sum,our work showed that excessive accumulation of Spt21 protein is toxic to cells and tightly control the level of Spt21 is of great significance for cell growth.This study reveals a new mechanism by which cell cycle regulates Spt21 protein stability.In G1 phase,Spt21 protein is degraded by ubiquitin-proteasome pathway.In S phase,Spt21 protein accumulates to activate the expression of histone genes.In G2 phase,Spt21 protein is degraded by autophagy pathway.These findings lay foundation for further study how Spt21 is degraded by autophagy.