染色质重塑复合体是一类利用ATP水解的能量来对核小体进行排布或者置换的蛋白复合物。它可以通过改变染色质的可接近性并协助特定反式作用因子结合到相应DNA序列从而调节基因表达。依据染色质重塑蛋白结构域特点可将它们分为4个家族，分别是SWI/SNF、ISWI、CHD和INO80家族。其中ISWI家族蛋白除N端共有的ATPase结构域外，在C端还含有HAND、SANT、SLIDE结构域。

在拟南芥中CHR11和CHR17为功能冗余的ISWI染色质重塑蛋白。有研究发现它们能够通过SLIDE结构域与含有DDT结构域蛋白结合，但并不清楚这些蛋白的具体组成方式和作用机理。本研究中我们首先通过亲和纯化结合质谱分析鉴定出几种ISWI复合体的亚型，它们分别是CRAF(CHR11/17-RLT1/2-ARID5-FHA2), CDM (CHR11/17-DDP1/2/3-MSI3), 和 CDD (CHR11/17-DDR1/3/4/5/DDW1)这三类复合体。在包含RLT1/2的CRAF复合体中，我们鉴定了两个新植物特异性亚基ARID5和FHA2。结合酵母双杂交，体内Co-IP以及体外Pull Down实验我们发现RLT1/2连接CHR11/17和ARID5、FHA2的结合，并且进一步确定了它们相互作用的方式。其中ARID5具有H3K4me3的识别能力，而FHA2对组蛋白变体H2A.Z具有亲和性。通过对以上几种基因突变体的转录组分析我们鉴定出了它们共同影响的基因。CHR11/17的突变对花的形态造成严重影响且产生不育表型，而其余几种突变体的育性均有不同程度的降低。有趣的是chr11/17、rlt/12、arid5三种突变体呈现早花表型，而fha2无早花表型，表明FHA2在复合体中出现功能的分化。FT、SEP3等开花调控基因的表达水平受fha2突变的影响远低于CRAF复合体其它几种组分的突变，这表明FHA2可能不参与对这些开花基因的转录调控。进一步研究发现FHA2在CRAF类染色质重塑复合体中的主要作用是维持花器官的正常发育以维持植物良好的育性。

此外，为了探究影响拟南芥ISWI蛋白重塑活性的因素，我们利用体外重构的核小体进行了核小体居中活性实验以及ATPase酶活实验，对可能影响拟南芥ISWI蛋白重塑活性的因素进行探索。结果表明RLT2蛋白具有抑制CHR11滑动核小体的功能。不同于其他物种的ISWI核心酶，CHR11/17可能缺少自抑制的AutoN结构域，我们推测RLT蛋白很可能起到协助抑制酶活的功能，并通过一系列辅助亚基将CHR11/17的活性保持在一个稳定的状态。这种通过辅助亚基调控ISWI酶活性可能是植物特异的一种调节模式。此外，存在于核小体的酸性基序对于CHR11发挥重塑功能也是不可或缺的。

Chromatin remodelers move or restructure nucleosomes using energy derived from ATP hydrolysis. By changing the accessibility of chromatin, the remodelers allow the trans-acting factor to bind and regulate gene transcription. There are four distinct families of chromatin remodeling complexes, namely SWI/SNF family, ISWI family, CHD family and INO80 family. All four remodelers utilize ATP hydrolysis to alter histone-DNA contacts and share a similar ATPase domain. Yet they are specialized for particular purposes and biological contexts, imparted by unique domains residing in their catalytic ATPases and also by their unique associated subunits. For ISWI family remodeler, a characteristic set of domains reside at its C terminus: HAND SANT and SLIDE domain.

ISWI proteins are conserved in eukaryotes and usually form complexes with DDT (DNA binding homeobox and different transcription factors) domain proteins. In Arabidopsis thaliana, ISWI (AtISWI) proteins are encoded by two functionally redundant genes: CHR11 and CHR17. In this study we systematically analyzed the constitution of AtISWI complex by affinity purification coupled with mass spectrometry. The AtISWI complexes are classified into three types. They are CRAF (CHR11/17-RLT1/2-ARID5-FHA2), CDM (CHR11/17-DDP1/2/3-MSI3) and CDD (CHR11/17-DDR1/3/4/5/DDW1). In addition, we identified two novel plant-specific components in the CRAF complex: ARID5 and FHA2. By Co-IP and in vitro pull-down assay, we confirmed that RLT1/2 act as linkers between CHR11/17 and ARID5 or FHA2. ARID5 is able to recognize the epigenetic marker H3K4me3, while FHA2 has high affinity towards the histone variant H2A.Z. By RNA-seq analysis, we identified genes that are differntially regulated in the mutants relative to the wild type. Mutations in CHR11/17 result in completely sterile flowers and aberrant floral organs. Loss-of function mutations of RLT1/2, ARID5, and FHA2 exhibit a reduced fertility. Interestingly the fha2 mutant does not have an early flower phenotype as the rlt1/2 and arid5 mutants, indicating that FHA2 may have a divergent role in AtISWI complex. We found that the flowering genes such as FT and SEP3 are upregulated in the rlt1/2 and arid5 mutant but not in the fha2 mutants, indicating that FHA2 is not involved in the regulation of flowering time. Further study revealed that FHA2 is exclusively involved in regulating floral morphogenesis and responsible for plant fertility.

Moreover, to explore the factors that regulate the remodeling activity of CHR11/17, we reconstituted the Nucleosome Core Particles from recombinant histones and DNA and performed a nucleosome centering assay and an ATPase assay. We found that RLT2 act as a brake in the process of CHR11 mediated nucleosome sliding. Unlike most of the eukaryotic ISWI, the AutoN domain is not conserved in Arabidopsis, we propose that the binding of RLT to CHR11 regulates the ATPase activity of AtISWI and may represent a plant-specific mechanism for the ISWI activity regulation. Additionally, we found the acidic patch in the histone H2A is indispensable for CHR11 to exert its nucleosome centering function.